The overall purpose of this procedure is to use microscopy as a tool to study the phagocytosis of the pathogenic fungal species Cryptococcus neoformans by its environmental predator amoeba. This video details a protocol for preparing a co-culture of cryptococcal cells and amoebae which I will study using confocal laser-scanning microscopy and transmission electron microscopy. Information obtained from using these techniques could shed insights into the behavior of cryptococcal cells when internalized during infection.
Streak out the test fungal strains on YPD agar plates. Incubate the plate for 48 hours at 30 degrees Celsius. Scrape off a loopful of cells from the 48-hour-old plate and inoculate them into a 250-milliliter conical flask containing 100 milliliters of YNB broth that is supplemented with 4%glucose.
Incubate the flask at 30 degrees Celsius for 24 hours on a rotary shaker. After a 24-hour incubation period, count the fungal cells using a hemocytometer and adjust the cell number to one million cells per milliliter with physiological buffer solution. Cultivate amoeba cells in peptone yeast extract glucose broth for a week at 30 degrees Celsius.
Count the amoeba cells using a hemocytometer and adjust the cell number to 10 million cells per milliliter with fresh sterile ATCC medium 712. Following this, perform a viability assay using a trypan blue stain. It is advisable to work with cells that show at least 80%viability.
Dispense a 200-microliter suspension of standardized amoeba cells into chambers of an adherent slide. Allow two hours to pass for cells to adhere to the surface at 30 degrees Celsius. While amoeba cells are settling down, stain the standardized cryptococcal cells with fluorescein isothiocyanate.
Gently agitate the fungal cells for two hours at room temperature and in the dark. Dispense 200-microliter suspension of stained cryptococcal cells after washing to appropriate chambers that already contain amoeba cells. Incubate the prepared co-culture at 30 degrees Celsius for an additional two-hour period.
At the end of the coincubation period, wash the chambers twice with PBS to remove any unbound amoeba cells including non-internalized cryptococcal cells. Aspirate the glutaraldehyde that was used to fix the cells, and wash the chamber twice with PBS. Dismantle the chamber slide.
View the co-cultured cells using a confocal laser-scanning microscope. To complement the above assay, stain the cryptococcal cells with pHrodo dye which selectively allows for the reading of cells trapped inside the food vacuole. Dispense these stained cells into the wells of a black adherent microtiter plate that already contains seeded amoeba cells.
Measure fluorescence on a microplate reader that can convert logarithmic signals to relative fluorescence units. Add five milliliters of standardized cryptococcal cells to the same centrifuge tube that already contains five milliliters of standardized amoeba cells. Allow the tube to stand for two hours at 30 degrees Celsius.
Aspirate the supernatant after centrifugation. Fix the pellet which represents the co-cultured cells with one molar of sodium phosphate buffered 3%glutaraldehyde at pH 7 for three hours. Wash the co-cultured cells once with sodium phosphate buffer.
Fix again for 1 1/2 hours in similarly buffered 1%osmium tetroxide followed by washing. Dehydrate the TEM material also known as the co-cultured cells in a graded acetone series of 30%50%70%95%and 100%for 15 minutes each. Here, repeat the final dehydration step in two changes.
Prepare the epoxy resin by weighing off epoxy of normal consistency. Embed material into the epoxy resin. Polymerize the TEM material for eight hours at 70 degrees Celsius.
Cut 16-nanometer sections with glass knives mounted on the ultratome. Stain the sections with uranyl acetate for 10 minutes in the dark followed by lead citrate for 10 minutes, also in the dark. View the sections with a transmission electron microscope.
For illustrative purposes, in the video, two isolates are examined, one that produces 3-hydroxy fatty acid and the other that does not. 3-hydroxy fatty acids are secondary metabolites that do not play role in the growth of cryptococcal cells. Here are snapshots that show interactive moments between Cryptococcus and amoebae.
To the point, a cryptococcal cell before and after internalization can be observed. When studying phagocytosis, it is ideal to make use of live imaging. However, it is not always possible for researchers to have access to such a microscope in order to follow the process of phagocytosis continuously.
A possible solution to compensate for this limitation may be to complement images with quantitative data through the reading of relative fluorescence units. The bar graph is an example of data that I used to complement the images. Importantly, both the qualitative data in the form of images and the quantitative data can be taken at different time points over the course of time.
By piecing all the information, one can start to construct a bigger image of what transpires during the process of phagocytosis. From the quantitative data, it is clear to see that the cells with 3-hydroxy fatty acids are less susceptible to amoeba action when compared to cells that do not produce 3-hydroxy fatty acid as fewer cells are internalized. The TEM is particularly a powerful tool as it can give a bird's-eye view into the lumen of the food vacuole because of its high resolution power, and this is the kind of detail that is often missed when examining live imaging as well as still images.
In this specific TEM micrograph, protuberances on the surface of cryptococcal cells can clearly be seen. Previously, it was theorized that these cell surface structures are channels by which 3-hydroxy fatty acids are released into the surrounding environment. To the point, these channels may help to deliver 3-hydroxy fatty acids into the lumen of the food vacuole of amoebae to alter the internal conditions, hence the promotion of cell survival.
pHrodo or like FITC may be a much better stain to use as it only fluoresce at a low pH, like inside the food vacuole. To be specific, pHrodo only allows the researcher to obtain data concerning internalized cells. Towards this end, it is important that cells are suspended in phosphate buffer solution before staining and that amoeba media is of a neutral pH.
Transmission electron microscope operators are to be well trained to section properly as this is often the point where the results could be distorted and micrograph damaged by electron bombardment. It is envisaged that researchers will be able to take enough information from the presented protocol and optimize them in their own studies. Moreover, researchers may also develop antibodies against targeted metabolite and apply them during their immunofluorescent microscopy study including immunogold labeling during TEM examination.
