We thank the Molecular Biosciences (MOBI) doctoral program and the Biology Department at Middle Tennessee State University (MTSU) for providing the funding for this study. The project was also funded in part by a Special Projects grant awarded to D.E.N. by the MTSU Foundation.

NOTE: C. neoformans is a Biosafety Level 2 (BSL-2) pathogen and researchers working with it must take proper precautions. Detailed procedures on how to safely work with BSL-2 pathogens can be found on the Center for Disease Control&#39;s (CDC) website, but it is important to note that all persons coming into contact with C. neoformans should be properly trained in handling pathogenic agents and should always wear appropriate personal protective equipment (PPE), generally latex or nitrile gloves. Further, rotors on centrifuges should be sealed to prevent aerosolization of samples and any spills cleaned up immediately using a 10% bleach solution28.
1. Capsule Induction
NOTE: All steps should be performed at room temperature unless otherwise noted.
<ol>
	<li>Culture C. neoformans in yeast peptone dextrose (YPD) broth (pH 6.5 +/-0.2) and incubate at 37 &#176;C with shaking until they are in log phase (approximately 18 - 36 h).</li>
	<li>Centrifuge cells at 870 x g for 5 min at 21 &#176;C and wash three times with phosphate buffered saline (PBS).</li>
	<li>Use a hemocytometer (or an automated cell counter) to count the cells and resuspend at 2 x 105 cells/2 mL in Dulbecco&#39;s minimal essential media (DMEM).</li>
	<li>To induce the capsule, incubate at 37 &#176;C and 5% CO2 for 18 h. 
	NOTE: The combination of the absence of nutrients in the media and the presence of CO2 stimulates C. neoformans capsule growth.</li>
	<li>Post-incubation, harvest the cells by centrifuging (as above) and removing all but 5-10 &#181;L supernatant. At the same time, harvest a corresponding un-induced cell sample for each strain to be measured. Use these as negative controls. 
	NOTE: The cell pellets will be very small and may be difficult to see. Be careful when aspirating supernatant.</li>
	<li>Stain with either India ink (Section 2.1) or fluorescent dyes (Section 2.2).</li>
</ol>
2. Staining
<ol>
	<li>Staining with only India ink
	<ol>
		<li>To stain the yeast with India ink, resuspend the pellet in the remaining supernatant and add 4&#956;L (approximately half) of cell suspension and 4&#956;L of India ink onto a glass microscope slide. Gently mix and avoid foaming.</li>
		<li>Apply a 13-mm glass coverslip (#1.5 thickness) and seal edges with a non-toxic sealant (clear nail polish) to prevent the sample from drying out.</li>
	</ol>
	</li>
	<li>Staining with only fluorescent dyes
	<ol>
		<li>To stain the yeast with a fluorescent dye, resuspend the cell pellet in 50 &#181;L PBS with 1% bovine serum albumin (BSA).</li>
		<li>Incubate the culture with an equal volume of Calcofluor white (CFW) (1 &#181;g/mL) and capsule mAb 18B7 (see Table of Materials) conjugated to AlexaFluor488 (AF488) (10 &#181;g/mL) for 30 min at 37 &#176;C.</li>
		<li>Centrifuge cells at 870 x g for 5 min at 21 &#176;C post-incubation and aspirate supernatant.</li>
		<li>Resuspend cell pellet in 10 &#181;L PBS with 1% bovine serum albumin (BSA).</li>
		<li>Pipette 8 &#181;L of cell suspension onto a glass microscope slide.</li>
		<li>Apply a 13-mm glass coverslip (#1.5 thickness) and seal edges with a non-toxic sealant (clear nail polish) to prevent the sample from drying out.</li>
	</ol>
	</li>
	<li>Staining with both India ink and fluorescent dyes
	<ol>
		<li>To more directly compare the efficacy of India ink staining to that of fluorescent dyes, co-stain the same cell population with both types of stain. To do so, first incubate cells with both a fluorescent capsule and cell wall stain, as per steps 2.2.1 - 2.2.3.</li>
		<li>Next, pipette equal volumes (4 &#181;L) of the fluorescently stained cell suspension and India ink onto a glass slide. Gently mix.</li>
		<li>Apply a 13-mm glass coverslip (#1.5 thickness) and seal the edges with a non-toxic sealant (clear nail polish) to prevent the sample from drying out.</li>
	</ol>
	</li>
</ol>
3. Image Acquisition/Microscopy
<ol>
	<li>Imaging cells stained with India ink.
	<ol>
		<li>Acquire images using a standard light microscope (100X magnification).</li>
		<li>Image a minimum of 50 cells per condition. 
		NOTE: The number of cells per field will vary, and this is acceptable. It is important, however, that overlapping cells be kept at a minimum, as these are difficult to measure (both manually and with automated software). For these experiments, images were acquired at a depth of 16 bits with exposure times optimized to maximize image contrast while minimizing the blur caused by small movements of the cells.</li>
	</ol>
	</li>
	<li>Imaging cells stained with fluorescent dyes
	<ol>
		<li>To image the cells by fluorescence microscopy, open the microscope imaging software and select the appropriate excitation and emission wavelengths for the fluorophores used (CFW and AF488 are excited using the 405nm and 488 nm lights, respectively). Acquire images using a fluorescence microscope.</li>
		<li>Image a minimum of 50 cells per condition. 
		NOTE: The number of cells per field will vary, and this is acceptable. It is important, however, that overlapping cells be kept at a minimum as these are difficult to measure (both manually and with automated software).</li>
		<li>Acquire a z-stack image series of the cells to ensure that the maximum capsule diameter for each cell within a given field is obtained.
		<ol>
			<li>In the microscope control software, click on the &#34;z-stack&#34; bar to open up the &#34;z-stack&#34; control panel, then select the &#34;First/Last&#34; option in the top left corner of the panel.</li>
			<li>Use the fine-focus control on the microscope to focus at a level that is below the widest point of a single yeast cell and the capsules for all within the current field of view and click &#8220;Set First&#8221;.</li>
			<li>Use the fine-focus control to focus above the widest point of the cell body of the same cell and of the capsule for all of the cells within the current field of view and click &#8220;Set Last&#8221;.</li>
			<li>Then, click &#8220;Start Experiment&#8221; on the &#8220;Acquisition&#8221; tab to acquire the z-stack for the current position. Repeat the process at multiple positions until z-stack images of a minimum of 50 cells have been acquired. 
			NOTE: For these experiments, the confocal pinhole was set to 1.0 AU and an overlapping z-series of 10-20 slices covering 0.7 &#181;M each were acquired at the selected positions. AU = Airy unit.</li>
		</ol>
		</li>
		<li>Next, convert each z-stack series into maximum intensity projections (MIP) using appropriate image analysis software. 
		NOTE: MIPs project the greatest intensity at every plane of the stacked image29. Creating MIPs allows one to produce a 2-D representation of images that correspond to the maximum cell and capsule diameter within the captured 3-D stack.</li>
	</ol>
	</li>
	<li>Imaging cells stained with both India ink and fluorescent dyes
	<ol>
		<li>Acquire images using either a widefield (40X magnification) or confocal microscope (63X or 100X magnification). For cells stained with both India ink and fluorescent dyes, capture images using a widefield microscope equipped with a computer-controlled stage and oil immersion objective. 
		NOTE: The microscope in use should be controlled using an imaging software CFW. fluorescence that is excited through a 340-380 nm excitation filter, and emitted light should be detected through a 435-485 nm barrier filter reflected from a 400-nm dichroic mirror. AF488 fluorescence can be excited through a 465-495 nm excitation filter, and emitted light can be detected through a 515-555 nm barrier filter reflected from a 505-nm dichroic mirror.</li>
		<li>Image a minimum of 50 cells per condition for each staining method.</li>
	</ol>
	</li>
</ol>
4. Manual Measurement of Capsule
<ol>
	<li>For cells stained with India ink or fluorescent stains, use a cell measurement software to manually measure capsule and cell diameter (see Table of Materials). To do this, go to &#34;File&#34; &#62; &#34;Open Image&#34; &#62; &#34;Measure&#34; and use the cursor to draw a straight line through the widest point of the whole cell (total diameter).</li>
	<li>Next, click &#34;Measure&#34; again and draw a straight line through the cell body (cell body diameter). 
	NOTE: Measurements are auto-saved onto the cells.</li>
	<li>Repeat this process for all cells (minimum of 50/group).</li>
	<li>To save images once they are measured, click &#34;File&#34; &#62; &#34;Save As&#34; and name the images appropriately.</li>
	<li>Select the newly measured files and export data to a spreadsheet software.</li>
	<li>Calculate the average capsule diameter by using the equation: ([total diameter] - [cell body diameter]).</li>
</ol>
5. Automated Measurement of Capsule
NOTE: For successful automated measurement, use 16-bit images with a high level of contrast and that are properly focused along with an appropriate image analysis software (see Table of Materials). When imaging C. neoformans for capsule measurement, it is important to focus on the dark ring at the boundary between the cell wall and capsule. This allows the software to correctly delineate the cell and capsule.
<ol>
	<li>Invert and create a copy of all India ink stained cell images using an appropriate image-editing software (see Table of Materials). To do this, click &#34;File&#34; &#62; &#34;Open&#34; to open India ink stained images and then &#34;Control&#34; + &#34;I&#34; to invert the image. Click &#34;File&#34; &#62; &#34;Save As&#34; to save the inverted image.</li>
	<li>To import both the original and inverted images into the software click &#34;File&#34; &#62; &#34;Import Sequence&#34; &#62; &#34;Load Image&#34; &#62; &#34;Define Sequence (Manually)&#34; &#62; &#34;Dimensions (Channel)&#34; &#62; &#34;Import&#34; &#62; &#34;Apply&#34;. 
	NOTE: The C. neoformans cell bodies and capsules can be identified and masked using specific algorithms - referred to as &#39;recipes&#39; - included in the software.</li>
	<li>Use the &#39;Colony Analyzer (Fluorescence)&#39; recipe on the inverted image to detect and mask the cell body, then click &#34;apply&#34;. In the inverted image, the dark ring defining the boundary of the C. neoformans cell becomes brighter than the surrounding capsule.</li>
	<li>Use the &#39;Colony Analyzer (Fluorescence)&#39; recipe on the inverted image to segment the C. neoformans cell bodies from their capsules, then click &#34;apply&#34;. This creates a count mask. Rename this count mask &#34;Cell Body&#34; by right-clicking the tab labeled &#34;count mask&#34;. 
	NOTE: The recipe consists of multiple image processing steps: background removal, object detection, object separation, and subset filtering.</li>
	<li>Next, use the &#39;Cell Proliferation&#39; recipe on the original image to detect and mask the capsule, and click &#34;apply&#34;. This creates a count mask. Rename this count mask &#34;Capsule&#34; by right-clicking the tab labeled &#34;count mask&#34;.</li>
	<li>Using the recipe, &#8216;Capsule Partition&#8217;, create a new mask onto the original image to partition adjacent capsules from one another. To do this, click &#8220;Capsule Partition&#8221; from the recipe dropdown menu. In the Capsule Partition Box, choose the now renamed count mask &#8220;Capsule&#8221; (5.5) for Capsule Region. Choose the now renamed count mask &#8220;Cell Body&#8221; for Crypto Cells (5.4). Choose &#8220;Original&#8221; for the input channel, and &#8220;Create Mask for Partitioned Capsule. Click &#8220;Apply 
	NOTE: The recipe generates cell and capsule diameter, defined as the average of the longest and shortest axes crossing the center of the cell and capsule, and area measurements from detection masks. Find the output that is located under the spreadsheet tab in this software. Repeat this process for all images (minimum of 50 cells/group). The recipe&#39;s parameters could be tuned to optimize detection of individual images or optimized for batch detection of multiple images. Additional measurements can be calculated by the recipe for comprehensive characterization of the cell and capsule&#39;s morphology.</li>
	<li>Export data to a spreadsheet software of choice for further analysis.</li>
</ol>

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</ol>

The author, Hoyin Lai, is the product manager at DRVision that publishes the automated image analysis software, Aivia.

For decades, the capsule has been a major focus of research for both mycologists and clinicians interested in C. neoformans and cryptococcosis due to its role as a major virulence factor for the pathogen. Using microscopy to measure differences in capsule size between strains and under different growth conditions can provide important information about the pathogen and its responses to various stimuli (i.e., different environmental conditions, potential drug treatments, etc.) Here, we have outl...

Affecting a quarter million people every year and resulting in more than 180,000 deaths annually, Cryptococcus neoformans is a pathogenic, intracellular yeast and the causative agent of cryptococcosis1,2,3. Hardest hit are HIV-positive patients in poor countries who do not have ready access to antiretroviral therapy, making them acutely susceptible to the illness4,5,6. Data from the CDC indicate that in sub-Saharan Africa, C. neoformans kills more people than tuberculosis annually and more every month than any Ebola outbreak on record1. The most common route of exposure occurs from inhaling desiccated spores that are commonplace in the environment7. Upon entering the lungs, there are several virulence factors that contribute to the success of C. neoformans within infected individuals. The polysaccharide capsule is considered the microbe&#39;s primary virulence factor, as acapsular strains are not virulent8.
The cryptococcal capsule is made of up three principle components: glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MPs)9. While MPs are a relatively minor cell wall-associated component of the capsule, they are immunogenic and can promote a mostly pro-inflammatory response9,10. In contrast, GXM and GalXM make up the bulk of the capsule (&#62;90% by weight) and have immunosuppressive effects11. In addition to its immunomodulatory effects, the rapid enlargement of the capsule in vivo creates a mechanical barrier to ingestion by host phagocytic cells (i.e., neutrophils and macrophages)12. The C. neoformans capsule and its synthesis are complex, but overall, increased capsule diameter is correlated with increased virulence6,13,14. Given this, it is important for C. neoformans researchers to be able to quickly and accurately quantify capsule measurements.
Both the C. neoformans cell and its polysaccharide capsule are dynamic structures and show changes over time15. The capsule can change in density, size, and assembly in response to changes in the host environment16,17,18. Low iron or nutrient levels, exposure to serum, the human physiological pH, and increased CO2 are known to initiate capsule growth16,18,19,20. Further, researchers have shown structural changes resulting in significant differences in immunoreactivity during an infection, lending an advantage to C. neoformans over its host21,22. This is known because the architecture of the C. neoformans capsule has been analyzed in a variety of ways. Electron microscopy, for example, has revealed that the capsule has a heterogeneous matrix with an inner electron-dense layer underneath an outer, more permeable layer23. Light scattering and the use of optical tweezers have allowed researchers to further elucidate its macromolecular properties24. Analyzing the results from both static and dynamic light scattering measurements, we know that the polysaccharide capsule has a complex branching structure23. Optical tweezers have been used to test the rigidity of the structure as well as evaluate its antibody reactivity24. However, by far the most frequently employed analysis of the C. neoformans capsule is the measurement of its size.
To quantify capsule size, researchers use what should be a simple measurement: the linear diameter of the capsule. Digital microscopes are used to capture images of multiple C. neoformans cells (generally hundreds) stained with either India ink or fluorescent dyes. The size of each cell body and surrounding capsule is measured. The data are compiled, and the average diameter of the capsule is calculated by subtracting the cell body diameter from the whole cell diameter (cell body + capsule). Up until this point, these measurements have been done manually. While generally accurate, this method has drawbacks for researchers. Large data sets can take days or even weeks to analyze by hand. And because these measurements are done manually, subjectivity and human error may affect the result.
Automated computational image analysis has become an indispensable tool for researchers in many areas of molecular cell biology, enabling faster and more reliable analysis of biological images 25,26,27. Precise image analysis techniques are necessary to mine quantitative information from what are often complex and immense data sets. However, some measurements, especially the measurement of C. neoformans capsule, have been difficult to automate. Accurately identifying the interface between the cell wall and capsule, which generally appears as a dark ring when imaged by phase-contrast microscopy, can be troublesome to resolve using a simple threshold. Further, C. neoformans cells in culture tend to clump together and accurate segmentation of the cells is necessary for accurate measurements.
The aim of this project was to (i) illustrate one of the standard protocols for capsule induction in C. neoformans, (ii) compare and contrast India ink and fluorescence staining as they pertain to capsule diameter measurements, (iii) develop simple, computational methods to measure capsule diameter using images of India ink stained cells using an image analysis software, and, (iv) assess the benefits and limitations of measuring capsule diameter manually and using software automation. We find that of the two staining methods, fluorescent labeling of the cell wall and capsule, while more time-consuming, provided the most consistent results between experiments. However, both methods enabled us to successfully distinguish between lab and clinical C. neoformans strains exhibiting different capsule sizes. Further, we were able to automate the measurement of capsule diameter from India ink stained images and found that this was a viable alternative to manual measurement of capsule.

<table><tbody><tr><td>&lt;strong&gt;Capsule Induction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;C. neoformans cells&lt;/em&gt;</td><td></td><td></td><td>The clinical lab strain, H99S, was a kind gift from Dr. John Perfect (Duke University).&amp;nbsp; The clinical strains, B18 and B52, were kind gifts from Dr. Greg Bisson (University of Pennsylania).&amp;nbsp;</td></tr><tr><td>Yeast Peptone Dextrose Broth (YPD)</td><td>Fisher Scientific</td><td>DF0428-17-5</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td></td><td></td><td>This is made in the lab using standard recipe (137mM NaCl, 2.7 mM KCl, 10mM Na2HPO4O, 2 mM Kh2PO4O)</td></tr><tr><td>DMEM/high-glucose with L-glutamine, without sodium pyruvate</td><td>GE Life Sciences</td><td>SH30022.01</td><td></td></tr><tr><td>6-well plates</td><td>Falcon</td><td>CL5335-5EA</td><td></td></tr><tr><td>Shaking incubator</td><td>Thermo Scientific&amp;nbsp;</td><td></td><td>MaxQ6000</td></tr><tr><td>CO2 incubator</td><td>Fisher Scientific</td><td></td><td>Isotemp</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td></td><td>Legend XTR</td></tr><tr><td>&lt;strong&gt;Staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td>Thermo Scientific</td><td></td><td>Legend Micro 21R</td></tr><tr><td>India ink</td><td>Fisher Scientific</td><td>14-910-56</td><td></td></tr><tr><td>Calcofluor white</td><td>Sigma-Aldrich</td><td>18909-100ML-F</td><td></td></tr><tr><td>18B7 mouse anti-GXM antibody conjugated to Alexafluor 488</td><td></td><td></td><td>A kind gift from Dr. Arturo Casadevall (Johns Hopkins University)&amp;nbsp;</td></tr><tr><td>PBS with 1% Bovine Serum Albumin (BSA)</td><td></td><td></td><td>PBS is the same recipe listed above (line 4) with 1% BSA added and filter sterilized.</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>Superfrost microscope slides</td><td>Fisher Scientific</td><td>12-550-143</td><td></td></tr><tr><td>Glass coverslips</td><td>Corning</td><td>2855-18</td><td>#1.5 thickness</td></tr><tr><td>Clear nail polish or other non-toxic sealant</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Image Acquisition&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Immersion oil</td><td>Cargille&amp;nbsp;</td><td>16484</td><td></td></tr><tr><td>Light microscope with immersion oil objective</td><td>Zeiss</td><td></td><td>Zeiss Axio A1 with a Plan - NEOFLUAR 100x oil immersion NA 1.30 objective</td></tr><tr><td>Light microscope camera</td><td>Zeiss</td><td></td><td>Zeiss Axiocam ErCD camera</td></tr><tr><td>Confocal microscope with oil immersion objective</td><td>Zeiss</td><td></td><td>LSM 700 laser scanning confocal equipped with a Plan-Apochromat 63X NA 1.4 oil immersion DIC M27 objective.&amp;nbsp;</td></tr><tr><td>Confocal microscope software</td><td>Zen 2009</td><td></td><td></td></tr><tr><td>Confocal microscope camera</td><td>Nikon</td><td></td><td>Nikon Ti-Eclipse with a Intensilight epifluorescence illuminator (Nikon), CoolSNAP MYO microscope camera (Photometrics), Plan Apo 60x NA 1.40 oil immersion objective (Nikon) and 1.5x magnification changer.&amp;nbsp;</td></tr><tr><td>Widefield imaging software</td><td>Nikon Elements (Nikon)</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Capsule Measurement&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image editing software</td><td>Photoshop (Adobe)</td><td></td><td></td></tr><tr><td>Microscope software for manual measurement</td><td>Axiovision (Carl Zeiss)</td><td></td><td></td></tr><tr><td>Image analysis software for automated meesurement</td><td>Aivia (DRVision Technologies)</td><td></td><td></td></tr><tr><td>Spreadsheet software</td><td>Excel (Microsoft)</td><td></td><td></td></tr></tbody></table>

size matters: measurement of capsule diameter in cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

To illustrate capsule induction, cell staining, imaging, and measuring techniques, we used three strains of C. neoformans: the common, well-characterized laboratory strain, H99S30, and two clinically isolated strains of previously unknown capsule diameter, B18 and B5231.
The workflow of capsule induction, staining, and image acquisition using India ink is shown in <strong ...

Watch this Scientific Journal Video about Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans at JoVE.com

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...

size-matters-measurement-capsule-diameter-cryptococcus

Research

JoVE Journal

Immunology and Infection

13.8K Views.  Middle Tennessee State University. De polysaccharide capsule is de primaire virulentiefactor in Cryptococcus neoformans, en haar grootte correleert met de virulentie van de stam. De diameter van de capsule metingen worden gebruikt bij het testen van de fenotypische en te meten van de therapeutische werking. Hier een standaardmethode van capsule inductie wordt gepresenteerd, en twee methoden van kleuring en het meten van de diameter worden vergeleken.

Video: Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are available to quantify and compare capsule production between different strains or mutants, there is no widely used method for sorting bacteria based on how much capsule they produce. We have developed a method to separate bacteria by capsule amount, using a discontinuous density gradient. This method is used to compare capsule amounts semi-quantitatively between cultures, to isolate mutants with altered capsule production, and to purify capsulated bacteria from complex samples. This method can also be coupled with transposon-insertion sequencing to identify genes involved in capsule regulation. Here, the method is demonstrated in detail, including how to optimize the gradient conditions for a new bacterial species or strain, and how to construct and run the density gradient.

We demonstrate the use of discontinuous density gradients to separate bacterial populations based on capsule production. This method is used to compare capsule amount between cultures, isolate mutants with a specific capsule phenotype, or to identify capsule regulators. Described here is the optimization and running of the assay.

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are ...

Genetics

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 ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

Bioengineering

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

Biology

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.

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.

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

Assessing the Putative Anticryptococcal Properties of Crude and Clarified Extracts from Mollusks

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The widespread use of antifungals in clinical settings, their use in agriculture, and strain hybridization have led to increased evolution of resistance. This rising rate of resistance against antifungals is a growing concern among clinicians and scientists worldwide, and there is heightened urgency to develop novel antifungal therapies. For instance, C. neoformans produces several virulence factors, including intra- and extra-cellular enzymes (e.g., peptidases) with roles in tissue degradation, cellular regulation, and nutrient acquisition. The disruption of such peptidase activity by inhibitors perturbs fungal growth and proliferation, suggesting this may be an important strategy for combating the pathogen. Importantly, invertebrates such as mollusks produce peptidase inhibitors with biomedical applications and anti-microbial activity, but they are underexplored in terms of their usage against fungal pathogens. In this protocol, a global extraction from mollusks was performed to isolate potential peptidase inhibitors in crude and clarified extracts, and their effects against classical cryptococcal virulence factors were assessed. This method supports the prioritization of mollusks with antifungal properties and provides opportunities for the discovery of anti-virulence agents by harnessing the natural inhibitors found in mollusks.

The human fungal pathogen Cryptococcus neoformans produces a variety of virulence factors (e.g., peptidases) to promote its survival within the host. Environmental niches represent a promising source of novel natural peptidase inhibitors. This protocol outlines the preparation of extracts from mollusks and the assessment of their effect on fungal virulence factor production.

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The ...

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

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