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.
