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, 2.0 mL/L chloroform) and 1 mL/L trace elements (trace elements: 40 mg/L Na2B4O7&#183;H2O, 400 mg/L CuSO4, 8 g/L ZnSO4, 800 mg/L MnSO4, 800 mg/L FePO4), adjust pH to 6.8 with 1 M NaOH, add 1 % (w/v) agar and the required supplements as described in16 and autoclave] (Figure 1). 
	NOTE: Salt solution, trace elements solution and supplements are autoclaved.</li>
	<li>Incubate for 2-3 days at 37 &#176;C.</li>
	<li>Use a sterile toothpick (or an inoculation loop), transfer a small number of conidia, by gently touching a single colony, to plates of complete media (CM) [CM: 10.0 g/L glucose, 2.0 g/L peptone, 1.0 g/L yeast extract, 1.0 g/L casamino acids, 20 mL/L salt solution, 1 mL/L trace elements, 5 mL/L vitamin solution (vitamin solution: 0.1 g/L riboflavin, 0.1 g/L nicotinamide, 0.01 g/L p-amino benzoic acid, 0.05 g/L pyridoxine HCl, 1.0 mg/L biotin), adjust pH to 6.8 with 1 M NaOH, add 1 % (w/v) agar and the required supplements as described in16 and autoclave]. Autoclave and store the vitamin solution in a dark bottle at 4 &#176;C. 
	NOTE: In case fungal growth is too dense to identify and isolate individual colonies, re-streak onto a new agar plate to obtain single colonies.</li>
	<li>Incubate for 3-4 days at 37 &#176;C.</li>
	<li>Obtain a conidial suspension of approximately 2 x 106 cells/mL by scratching 1 cm from the surface of a conidiated fungal colony grown on CM agar plates, using a sterile toothpick (Figure S1). 
	NOTE: When necessary, count conidia with a hemocytometer.</li>
	<li>Harvest conidia of A. nidulans in a sterile 1.5 mL centrifuge tube with 1.0 mL of autoclaved distilled water containing 0.05% (v/v) Tween 80 for reducing the number of conidia clumps. 
	NOTE: Conidia can be stored for up to 2-3 weeks at 4 &#176;C without a relevant loss of viability (A. Athanasopoulos and V. Sophianopoulou, unpublished data). However, it is recommended to filter and/or to wash conidial suspension to remove mycelial parts and nutrients, in order to prevent conidial swelling.</li>
</ol>
2. Preparation for imaging filamentous fungi growing on agar (solid) mediums
NOTE: A modified version of the &#39;inverted agar method17,18 is used.
<ol>
	<li>Initially, spot 10 &#181;L aliquots of vigorously vortexed conidial (approximately 2 x 104 cells/mL) at several points onto Petri dishes (&#216;9 cm) 15 mL of MM with 1% (w/v) agar (Figure 2). 
	NOTE: Using the modified version of the &#39;inverted agar method&#39;, it is possible to image fungal samples for many hours without apparent deleterious effects on growing hyphae.</li>
	<li>Incubate the experimental culture according to the developmental stage intended to be investigated.</li>
	<li>Slice out a &#8776;0.8 mm2 block of agar containing the colony using a sterile scalpel. 
	NOTE: The dimensions of the agar block to be sliced out depends on the dimensions of the equipment to be placed afterwards. In the present work, 8 well &#181;-slides are used (see below).</li>
	<li>Invert and place the agar block into a well of an &#181;-slide or similar 8 chambered coverglass with coverslip suitable for live imaging. 
	&#8203;NOTE: In case transmitted light microscopy will be used in combination with fluorescence (labeling) microscopy, the agar block can be inverted onto a droplet of liquid medium containing the live cell staining dye, just before imaging.</li>
</ol>
3. Preparation for imaging filamentous fungi growing on liquid medium
<ol>
	<li>Transfer 10 &#181;L aliquots of a vigorously vortexed conidial suspension (approximately 2 x 104 cells/mL) in the wells of an 8 well &#181;-slide containing 200 &#181;L of (liquid) MM with the appropriate supplements (see above).</li>
	<li>Incubate for the desired time at the desired temperature (Figure 3). 
	&#8203;NOTE: If transmitted light microscopy will be used in combination with fluorescence (labeling) microscopy, liquid cultures have the great advantage that fluorescent dyes can be added at any desired time point during the experiment19.</li>
</ol>
4. Capture images
NOTE: The choice of microscope depends upon the available equipment. In any case the microscope setup should include an inverted stage, an environmental chamber or at least a room with precise air temperature control.
<ol>
	<li>Preheat the thermostated microscope chamber at 37 &#176;C (unless otherwise indicated or as suitable for the used fungal species) to stabilize the temperature before starting. This chamber allows temperature modulation of the microscope optics and sample stage during time-lapse experiments. Be aware that optical aberrations20 are introduced when normal immersion oils (designed for use at 23 &#176;C) oils are used at 37 &#176;C or above. 
	NOTE: On a tight budget, incubation chambers can be made out of cardboard and insulating packing material21 or by using a 3D printer22.</li>
	<li>Turn on the microscope, the scanner power, the laser power and computer, and load the imaging software. Place the &#181;-slide (prepared previously) in the microscope stage and focus.</li>
	<li>Find fields of view that contain isolated/not overlapping cells (or at least not overcrowded), in order to facilitate growth measurements during image analysis. Capture at least 50 growing cells per sample&#160;to allow robust statistical analysis.</li>
	<li>Select the desired transmitted light microscopy approach. Reduce the exposure time or laser power and pixel dwell time and/or increase pinhole diameters, in order to minimize photobleaching of fungal cells, as described elsewhere 23,24.</li>
	<li>Set microscope to acquire images at desired time intervals and start time series acquisition. 
	&#8203;NOTE: To correct for focal drift over time (especially for long experiments), due to thermal drift, diverse cell sizes, and cell motion use an autofocus strategy if&#160;available in your microscope software.</li>
</ol>
5. Image Analysis
NOTE: This section describes the key steps of processing time-lapse microscopy images for measuring growth rate of A. nidulans. Opening, visualization and processing of images is accomplished with the open source ImageJ/Fiji software25.
<ol>
	<li>Import the images to Fiji using Plugins | Bio-Formats | Bio-Formats Importer from the Fiji menu with default settings (Figure 4A). 
	NOTE: Check whether the Bio-Formats Importer properly recognize the image calibration. The picture dimension shown in the upper information field image window, must be equal to the original picture dimensions (Figure 4B). Press Shift + P to display and change image properties in ImageJ/Fiji software.</li>
	<li>Where needed, use histogram matching26 for illumination correction between different frames (Image | Adjust | Bleach Correction | Histogram Matching) (Figure 4C).</li>
	<li>Where needed, use SIFT-algorithm (Plugins | Registration | Linear Stack Alignment with SIFT) for aligning or matching image stacks (Figure 4D). Selecting &#34;Translation&#34; from the expected transformation menu should be enough to correct any x-y drift. 
	NOTE: Other plugins can be also used to align a stack of image slices, such as Image Stabilizer (https://imagej.net/Image_Stabilizer)&#160;or StackReg (http://bigwww.epfl.ch/thevenaz/stackreg/).</li>
	<li>Select hyphae that grow parallel to coverslip, avoiding those that are tilted. Be sure to select hyphae that propagate by polar extension and avoid hyphae presenting lateral and/or apical branching.</li>
	<li>Use MTrackJ (Plugins | MTrackJ) plugin to track growing hyphal tips (Figure 4E)27. To add a track, select the Add button in the toolbar and place the first point at a 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 Esc key) (Figure 4F). Move to another point of interest (i.e., growing hyphal tip) in the initial time frame and restart the procedure by measuring growth rate of another hypha. 
	NOTE: To install MTrackJ follow the instructions presented at https://imagescience.org/meijering/software/mtrackj/</li>
	<li>Click the Measure button in the MTrackJ dialog (Figure 4G) to open the output table. Save track measurements (File | Save As) to the desired file format (e.g., csv), analyze and plot them (Figure 4H). 
	NOTE: By selecting the Movie button, a movie is produced showing the image and track progression.</li>
</ol>

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<li>Barry, D. J., Williams, G. A., Chan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+analysis+of+filamentous+microbial+morphology+with+AnaMorf%5BTitle%5D)+AND+%22Biotechnology+Progress%22%5BJournal%5D)">Automated analysis of filamentous microbial morphology with AnaMorf.</a> Biotechnology Progress. 31 (3), 849-852 (2015).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>&amp;micro;-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&amp;Delta; ngnA&amp;Delta;</td><td></td><td></td><td>Genotype: zhA&amp;Delta;::pyrGAf; ngnA&amp;Delta;::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 &amp;micro;L</td><td>KISKER</td><td>VL004G</td><td></td></tr><tr><td>Quali - Standard Tips, Bevelled, 1-200 &amp;micro;L</td><td>KISKER</td><td>VL700G</td><td></td></tr><tr><td>Quali Microvolume Tips, DNase-, RNase free, 0,1-10 &amp;micro;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&amp;Delta;::argB+&amp;nbsp; 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&amp;Delta;::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>ZnSO&lt;sub&gt;4&lt;/sub&gt;</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 ...

quantitative-analysis-aspergillus-nidulans-growth-rate-using-live

Research

JoVE Journal

Biology

3.7K Views.  National Centre for Scientific Research, Demokritos (NCSRD). In deze video beschrijven we een labelvrij live imaging protocol om beelden van de modelschimmel aspergillus nidulans vast te leggen, met behulp van microscopietechnieken voor doorgegeven licht. We gebruiken deze beelden om de groeikinetiek van aspergillus nidulans in zowel vloeibare als vaste media te analyseren en te kwantificeren. Een visuele weergave van dit protocol stelt gebruikers in staat om vertrouwd te raken met belangrijke stappen van het vastleggen van afbeeldingen en microscopie ...

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

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

The filamentous fungus A. niger is a widely used strain in a broad range of industrial processes from food to pharmaceutical industry. One of the most intriguing and often uncontrollable characteristics of this filamentous organism is its complex morphology. It ranges from dense spherical pellets to viscous mycelia (Figure 1). Various process parameters and ingredients are known to influence fungal morphology 1. Since optimal productivity correlates strongly with a specific morphological form, the fungal morphology often represents the bottleneck of productivity in industrial production.
A straight forward and elegant approach to precisely control morphological shape is the addition of inorganic insoluble micro particles (like hydrous magnesium silicate, aluminum oxide or titanium silicate oxide) to the culture medium contributing to increased enzyme production 2-6. Since there is an obvious correlation between micro particle dependent morphology and enzyme production it is desirable to mathematically link productivity and morphological appearance. Therefore a quantitative precise and holistic morphological description is targeted.
Thus, we present a method to generate and characterize micro particle dependent morphological structures and to correlate fungal morphology with productivity (Figure 1) which possibly contributes to a better understanding of the morphogenesis of filamentous microorganisms.
The recombinant strain A. niger SKAn1015 is cultivated for 72 h in a 3 L stirred tank bioreactor. By addition of talc micro particles in concentrations of 1 g/L, 3 g/L and 10 g/L prior to inoculation a variety of morphological structures is reproducibly generated. Sterile samples are taken after 24, 48 and 72 hours for determination of growth progress and activity of the produced enzyme. The formed product is the high-value enzyme &beta;-fructofuranosidase, an important biocatalyst for neo-sugar formation in food or pharmaceutical industry, which catalyzes among others the reaction of sucrose to glucose 7-9. Therefore, the quantification of glucose after adding sucrose implies the amount of produced &beta;-fructofuranosidase. Glucose quantification is made by a GOD/POD-Assay 10, which is modified for high-throughput analysis in 96-well micro titer plates.
Fungal morphology after 72 hours is examined by microscope and characterized by digital image analysis. In doing so, particle shape factors for fungal macro morphology like Feret's diameter, projected area, perimeter, circularity, aspect ratio, roundness und solidity are calculated with the open source image processing program ImageJ. Relevant parameters are combined to a dimensionless Morphology number (Mn) 11, which enables a comprehensive characterization of fungal morphology. The close correlation of the Morphology number and productivity are highlighted by mathematical regression.

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

A method to precisely generate and to comprehensively characterize morphology of filamentous fungus Aspergillus niger is described, which allows the mathematical correlation of morphological appearance and productivity.

The filamentous fungus A. niger is a widely used strain in a broad range of industrial processes from food to pharmaceutical industry. One of the most ...

Immunology and Infection

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

ScanLag: High-throughput Quantification of Colony Growth and Lag Time

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are affected both by the doubling time of the microorganism and by any delay in growth upon transfer from one condition to another, the lag. The ScanLag method enables the characterization of these two independent properties at the level of colonies originating each from a single cell, generating a two-dimensional distribution of the lag time and of the growth time. In ScanLag, measurement of the time it takes for colonies on conventional nutrient agar plates to be detected is automated on an array of commercial scanners controlled by an in house application. Petri dishes are placed on the scanners, and the application acquires images periodically. Automated analysis of colony growth is then done by an application that returns the appearance time and growth rate of each colony. Other parameters, such as the shape, texture and color of the colony, can be extracted for multidimensional mapping of sub-populations of cells. Finally, the method enables the retrieval of rare variants with specific growth phenotypes for further characterization. The technique could be applied in bacteriology for the identification of long lag that can cause persistence to antibiotics, as well as a general low cost technique for phenotypic screens.

ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and immunological tolerance, while in immunocompromised patients, conidia can colonize airways and cause severe invasive respiratory disorders. Various cells in different airway compartments are involved in the immune response that prevents fungal invasion; however, the spatio-temporal aspects of pathogen elimination are still not completely understood. Three-dimensional (3D) imaging of optically cleared whole-mount organs, particularly the lungs of experimental mice, permits detection of fluorescently labeled pathogens in the airways at different time points after infection. In the present study, we describe an experimental setup to perform a quantitative analysis of A. fumigatus conidia distribution in the airways. Using fluorescent confocal laser scanning microscopy (CLSM), we traced the location of fluorescently labeled&#160;conidia in the bronchial branches and the alveolar compartment 6 hours after oropharyngeal application to mice. The approach described here was previously used for detection of the precise pathogen location and identification of the pathogen-interacting cells at different phases of the immune response. The experimental setup can be used to estimate the kinetics of the pathogen elimination in different pathological conditions.

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 &#181;m in size) in the airways of mice. The method also can be used for the analysis of microparticles and nanoparticle agglomerate distribution in the airways in various pathological condition models.

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The fermentable carbon concentration mainly governs how the yeast cells generate ATP; thus, the variation in fermentable carbohydrate levels drives the energetic metabolism of S. cerevisiae. This paper describes a high-throughput method based on exponential yeast growth to estimate the effects of concentration changes and nature of the carbon source on respiratory and fermentative metabolism. The growth of S. cerevisiae is measured in a microplate or shaken conical flask by determining the optical density (OD) at 600 nm. Then, a growth curve is built by plotting OD versus time, which allows identification and selection of the exponential phase, and is fitted with the exponential growth equation to obtain kinetic parameters. Low specific growth rates with higher doubling times generally represent a respiratory growth. Conversely, higher specific growth rates with lower doubling times indicate fermentative growth. Threshold values of doubling time and specific growth rate are estimated using well-known respiratory or fermentative conditions, such as non-fermentable carbon sources or higher concentrations of fermentable sugars. This is obtained for each specific strain. Finally, the calculated kinetic parameters are compared with the threshold values to establish whether the yeast shows fermentative and/or respiratory growth. The advantage of this method is its relative simplicity for understanding the effects of a substance/compound on fermentative or respiratory metabolism. It is important to highlight that growth is an intricate and complex biological process; therefore, preliminary data from this method must be corroborated by the quantification of oxygen consumption and accumulation of fermentation byproducts. Thereby, this technique can be used as a preliminary screening of compounds/substances that may disturb or enhance fermentative or respiratory metabolism.

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Here we present a protocol to estimate the respiratory and fermentative metabolism by fitting the exponential growth of Saccharomyces cerevisiae to the exponential growth equation. Calculation of the kinetic parameters allows for the screening of influences of substances/compounds on fermentation or mitochondrial respiration.

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The ...

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

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