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...

<ol>
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L., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+life+of+Cryptococcus+neoformans.">The intracellular life of Cryptococcus neoformans.</a> Annu Rev Pathol. 9, 219-238 (2014).</li><li>Rajasingham, R., 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=Global+burden+of+disease+of+HIV-associated+cryptococcal+meningitis:+an+updated+analysis.">Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis.</a> Lancet Infect Dis. 17 (8), 873-881 (2017).</li><li>Limper, A. H., Adenis, A., Le, T., Harrison, T. 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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 border="0" cellpadding="0" cellspacing="0" width="983">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">Capsule Induction</td>
			<td style="width:204px;"></td>
			<td style="width:117px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C. neoformans cells</td>
			<td></td>
			<td></td>
			<td>The clinical lab strain, H99S, was a kind gift from Dr. John Perfect (Duke University).&#160; The clinical strains, B18 and B52, were kind gifts from Dr. Greg Bisson (University of Pennsylania).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Peptone Dextrose Broth (YPD)</td>
			<td>Fisher Scientific</td>
			<td>DF0428-17-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">DMEM/high-glucose with L-glutamine, without sodium pyruvate</td>
			<td>GE Life Sciences</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plates</td>
			<td>Falcon</td>
			<td>CL5335-5EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaking incubator</td>
			<td>Thermo Scientific&#160;</td>
			<td></td>
			<td>MaxQ6000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 incubator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Isotemp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Legend XTR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Legend Micro 21R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">India ink</td>
			<td>Fisher Scientific</td>
			<td>14-910-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcofluor white</td>
			<td>Sigma-Aldrich</td>
			<td>18909-100ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips</td>
			<td>Corning</td>
			<td>2855-18</td>
			<td>#1.5 thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clear nail polish or other non-toxic sealant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image Acquisition&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersion oil</td>
			<td>Cargille&#160;</td>
			<td>16484</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Light microscope camera</td>
			<td>Zeiss</td>
			<td></td>
			<td>Zeiss Axiocam ErCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope software</td>
			<td>Zen 2009</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Widefield imaging software</td>
			<td>Nikon Elements (Nikon)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capsule Measurement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image editing software</td>
			<td>Photoshop (Adobe)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope software for manual measurement</td>
			<td>Axiovision (Carl Zeiss)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image analysis software for automated meesurement</td>
			<td>Aivia (DRVision Technologies)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

The overall goal of this procedure is to stain the polysaccharide capsule of C.neoformans strains for the visualization and measurement of the capsule diameter. These methods can help researchers with the induction, imaging, and measurement of capsule size in Cryptococcus neoformans. The main advantage of the staining and imaging techniques is that they are simple, fast, and allow for an accurate measurement of capsule diameter.Visual demonstration of this method is critical as the software steps may be confusing the first time it is used. Diameter of the Cryptococcus neoformans'capsule is often used as a measure of virulence. The ability to automate this process can save significant analysis time.To induce capsule production, first incubate a C.neoformans colony in yeast peptone dextrose broth at 37 degrees Celsius with shaking for 18-36 hours until the culture is in log phase. At the end of the culture, pellet the yeast by centrifugation and wash the yeast three times in PBS. After the last wash, resuspend the yeast at two times 10 to the fifth cells per two milliliters of DMEM concentration and incubate the culture at 37 degrees Celsius and 5%carbon dioxide for 18 hours.The next day, collect the cells by centrifugation and remove all but the last 10 microliters of the supernatant. To stain the yeast with India ink, resuspend the pellet in the remaining supernatant and transfer four microliters of the cell suspension and four microliters of India ink onto a glass microscope slide. Gently mix the resulting solution without creating bubbles.Then place a number 1.5 thickness 13 millimeter glass coverslip over the cell sample and seal the edges with a nontoxic sealant. To stain the yeast with a fluorescent dye, resuspend the overnight culture pellet in 50 microliters of PBS supplemented with 1%bovine serum and incubate the cells in 55-60 microliters of calcofluor-white and Alexa Fluor 488 conjugated capsule monoclonal 18B7 antibody for 30 minutes at 37 degrees Celsius. At the end of the incubation, collect the cells by centrifugation and resuspend the pellet in 10 microliters of PBS plus bovine serum albumin.Then transfer eight microliters of cells onto a glass microscope slide, cover the cell sample with a number 1.5 thickness 13 millimeter glass coverslip and seal the edges with a nontoxic sealant. To stain the yeast with both dyes, first incubate the cells with a fluorescent capsule and cell wall stain as just demonstrated. Then add four microliters of fluorescently-stained cells and four microliters of India ink onto a glass slide and place a sealed coverslip over the fungal cell sample as demonstrated.To image the cells by light microscopy, select the 100X objective and image at least 50 non-overlapping cells per condition with exposure times optimized to maximize the image contrast while minimizing the blur caused by small cell movements. To image the cells by fluorescence microscopy, open the microscope imaging software and select the appropriate excitation and emission wavelengths for the fluorophores used. In the Z stack control panel, select the first last option.Using the fine focus control on the microscope, first focus below the widest point of the cell body of a single yeast cell and the capsules for all of the cells within the current field of view and click set first. Next, 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 set last. Then open the acquisition tab and click start experiment to acquire the Z stack for the current position.To manually measure the capsule and cell diameters, open an appropriate cell measurement program and select the image to be measured. Select measure and use the cursor to draw a straight line through the widest point of the whole cell to measure the total cell diameter. Click measure again and draw a straight line through the cell body to measure the cell body diameter and open the next cell.When all of the cells have been measured, save the images. For automated measurement of the capsule and cell diameters, first open an appropriate image editing program and invert all of the India ink stained cell images. Next, to import both the original and inverted images into the cell measurement software, open the file menu in the measurement program and select import sequence, load image, define sequence manually, dimensions channel, import, and apply.To detect and mask the cell body, select the colony analyzer fluorescence protocol for the first inverted image of interest and click apply. Select the colony analyzer fluorescence protocol again to segment the inverted C.neoformans cell body images from their capsules and click apply again to create a count mask. Right click to rename the count mask cell body and use the cell proliferation protocol on the original image to detect and mask the capsule.Click apply to create a count mask for the original image and rename the count mask capsule. To create a new mask over the original image to partition the adjacent capsules from one another, select capsule partition and capsule for the capsule region. To export the data, click on spreadsheet then click on the export to spreadsheet icon.Select the measurements that you wish to export and then finally save the data. A capsule size increase is apparent in most strains post-induction, although some strains exhibit naturally small capsules. In this representative experiment, a significant difference in capsule size was consistently observed between the largest strain and the two smaller strains.Differences in capsule size can also be detected using fluorescent dyes. Z stack image capture ensures that the maximum capsule and cell body diameter is acquired for each cell. The stacks can then be processed to produce maximum intensity projections for the manual measurement of the yeast cell capsule and cell diameter.There is no significant intra-experiment difference between capsule measurements using either staining method, although a greater inter-experiment variability is observed when using India ink to measure capsule diameters compared to fluorescent dye staining. Significant differences in capsule diameter measurements between different researchers are not typically observed regardless of the method used while small but significant differences in the capsule size can be detected using the automated measurement method. For successful automated measuring, the cells must be in focus, have a medium to large capsule, and not be overly clustered.After watching this video, you should have a good understanding of how to induce the capsule in Cryptococcus neoformans, stain it using either India ink or fluorescent staining, image the capsule, and measure it via automated software.

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Research

JoVE Journal

Immunology and Infection

13.4K visualizações.  Middle Tennessee State University. The overall goal of this procedure is to stain the polysaccharide capsule of C.neoformans strains for the visualization and measurement of the capsule diameter. These methods can help researchers with the induction, imaging, and measurement of capsule size in Cryptococcus neoformans. The main advantage of the staining and imaging techniques is that they are simple, fast, and allow for an accurate measurement of capsule diameter.Visual demonstration of this method is critical as the sof...