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Method Article

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

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DOI:

10.3791/58698

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June 22nd, 2019

 , 
1Department of Microbial, Biochemical and Food Biotechnology, University of the Free State

In This Article

Summary

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.

Abstract

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.

Introduction

Microbes have evolved over time to occupy and thrive in different ecological niches such as the open physical boundaries of the soil and water, among others1. In these niches, microbes often engage in the direct competition for limited resources; importantly, for nutrients that they use for supporting their growth or space, which they need to accommodate the expanding population2,3. In certain instances, some holozoic organisms like amoeba may even predate on cryptococcal cells as a way of extracting nutrients from their biomass4,5. In turn, this allows such organisms to establish territorial dominance via controlling the population numbers of its prey. Because of this predatory pressure, some prey may be selected to produce microbial factors, such as the cryptococcal capsule6, to reconcile the negative effects of the pressure. However, as an unintended consequence of this pressure, some microbes acquire factors that allow them to cross the species barrier and seek out new niches to colonize7, like the confined spaces of the human body that are rich in nutrients and have ideal conditions. The latter may explain how a terrestrial microbe like Cryptococcus (C.) neoformans can transform to become pathogenic.

To this end, it is important to study the initial contact that cryptococcal cells may have with amoeba and how this may select them to become pathogenic. More specifically, this may give clues on how cryptococcal cells behave when acted upon by macrophages during infection. It is for this reason that amoeba was chosen as a model for macrophages here, as it is relatively cheap and easy to maintain a culture of amoeba in a laboratory8. Of interest was to also examine how cryptococcal secondary metabolites viz. 3-hydroxy fatty acids9,10 influence the interaction between amoebae and cryptococcal cells.

A simple way of perceiving the interaction between amoeba and its prey with the naked eye is to create a lawn using its prey on the surface of an agar plate and spot amoeba. The visualization of plaques or clear zones on the agar plate depicts areas where amoeba may have fed on its prey. However, at this macro level, only the outcome of the process is noted, and the process of phagocytosis is mechanized cannot be observed. Therefore, to appreciate the process on a cell-to-cell basis, there are several microscopic methods that can be used11,12. For example, an inverted microscope with an incubation chamber can be used to video record a time-lapse of events between a phagocytic cell and its target13. Unfortunately, due to the cost of a microscope with a time-lapse functionality, it is not always possible for laboratories to purchase such a microscope, especially in resource poor-settings.

To circumvent the above limitation, this study presents a sequential exploratory design that evaluates the interaction of C. neoformans viz C. neoformans UOFS Y-1378 and C. neoformans LMPE 046 with Acanthamoeba castellani. First, a qualitative method is used that precedes a quantitative method. Still images are captured using an inverted fluorescence microscope, as well as a transmission electron microscope to depict amoeba-Cryptococcus interactions. This was followed by quantifying fluorescence using a plate reader to estimate the efficiency of amoeba to internalize cryptococcal cells. When reconciling findings from these methods during the data-interpretation stage, this may equally reveal as much critical information as perusing a phagocytosis time-lapse video.

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Protocol

Cryptococcus neoformans and some Acanthamoeba castellanii strains are regarded as biosafety level-2 (BSL-2) pathogens; thus, researchers must take proper precautions when working with these organisms. For example, laboratory personnel should have specific training and personal protective equipment (PPE) such as lab coats, gloves, and eye protection. A biological safety cabinet (level-2) should be used for procedures that can cause infection14.

1. Cultivation and standardization of fungal cells (modified from Madu et al. 15 )

  1. Streak out the test fungal strains (i.e., C. neoformans UOFS Y-1378 and C. neoformans LMPE 046) from stock cultures (no older than 9 months) on yeast-peptone-dextrose (YPD) agar plates. Information on YPD agar's ingredients can be found in Table 1.
    NOTE: C. neoformans UOFS Y-1378 has been shown to produce 3-hydroxy fatty acids, while C. neoformans LMPE 046 does not produce 3-hydroxy fatty acids. Refer to Supplemental File 1 for information on how the presence of these molecules is determined.
  2. Incubate the agar plates for 48 h at 30 °C.
    NOTE: A plate can be stored for up to 2 months at 4 °C before it can be discarded or used to make a stock culture16.
  3. Scrape off a loopful of cryptococcal cells (C. neoformans UOFS Y-1378 or C. neoformans LMPE 046) from the 48 h-old plate and inoculate into a 250 mL conical flask containing 100 mL of the chemically-defined YNB broth (6.7 g/L) supplemented with 4% (w/v) glucose. Information on YNB broth's ingredients can be found in Table 2.
  4. Incubate the flasks at 30 °C for 24 h while agitating at 160 rpm on a rotary shaker.
  5. After a 24 h incubation period, count the fungal cells using a hemocytometer and adjust the cell number to 1 x 106 cells/mL with PBS at pH 7.4.
    NOTE: The prepared C. neoformans UOFS Y-1378 inoculum was used in steps 3.1 and 3.2, while C. neoformans LMPE 046 inoculum was only used in step 3.2.

2. Cultivation and standardization of amoeba cells (modified from Madu et al. 15 )

  1. Thaw a stock culture of Acanthamoeba castellanii and bring it to room temperature (RT).
    NOTE: Amoeba was prepared based on the modified protocols of Axelsson-Olsson et al.8 and Schuster17.
  2. Pipette 1 mL of the thawed culture and inoculate it into a 50 mL centrifuge tube containing 15 mL of ATCC medium 712. Information on ATCC medium 712's ingredients can be found in Table 3.
  3. Manually shake it gently and immediately centrifuge for 5 min at 400 x g and 30 °C.
  4. Aspirate the supernatant.
  5. Resuspend the cells in 15 mL of ATCC medium 712 and incubate the tube at 30 °C for 14 days.
    NOTE: Periodically check the cells, using a simple light microscope, to determine if they are in a trophozoite state. Once they are in a trophozoite state, start a fresh culture.
  6. Pipette 1 mL from a culture that shows cells in a trophozoite state and use it to inoculate a sterile 50 mL centrifuge tube containing 15 mL of fresh, sterile ATCC medium 712.
  7. Incubate the tube at 30 °C for 1 week while agitating at 160 rpm on a rotary shaker.
  8. After a week, count the amoeba cells using a hemocytometer and adjust the cell number to 1 x 107 cells/mL with fresh, sterile ATCC medium 712.
  9. Perform a viability assay using a trypan blue stain as detailed by Strober18. Proceed further with the cultures that show at least 80% viability.

3. Fluorescence staining of cells to study phagocytosis (modified from Madu et al. 15 )

  1. Gathering qualitative data through use of fluorescence microscope
    NOTE: Perform this assay with Acanthamoeba castellanii and C. neoformans UOFS Y-1378.
    1. Dispense a 200-µL suspension of standardized amoebae (1 x 107 cells/mL in ATCC medium 712) into chamber wells of an adherent slide and incubate for 2 h at 30 °C for cells to adhere to the surface.
    2. While amoeba cells are settling down to adhere, stain the standardized C. neoformans UOFS Y-1378 cells that were adjusted to 1 x 106 cells/mL (in 999 µL of PBS) with 1 µL of fluorescein isothiocyanate in a 1.5 mL plastic tube.
      NOTE: Prepare the stain by dissolving 1 mg of fluorescein isothiocyanate in 1 mL of acetone.
    3. Gently agitate the C. neoformans UOFS Y-1378 cells on an orbital shaker set at 50 rpm for 2 h at RT and in the dark.
    4. After 2 h, centrifuge at 960 x g for 5 min at 30 °C to pellet the cells.
    5. Aspirate the supernatant to remove the PBS with the stain.
    6. Add 1 mL of PBS to the tube for washing the cell pellet. Wash the cells by gently pipetting.
    7. Centrifuge the cells at 960 x g for 5 min at 30 °C. Discard the supernatant. Repeat the washing step one more time.
    8. Resuspend the washed cells in 1 mL of PBS.
    9. Dispense a 200 µL suspension of the stained C. neoformans UOFS Y-1378 cells to chamber wells containing the unstained amoeba cells.
    10. Incubate the prepared co-culture at 30 °C for an additional 2 h period.
      NOTE: The co-culture can be incubated for different time points to suit the purpose of the experiment.
    11. At the end of the co-incubation period, aspirate the contents of the wells.
    12. Add 300 µL of PBS to the wells to wash the chamber wells and to remove any unbound co-cultured cells. Do this by gentle pipetting. Aspirate the contents of the wells. Repeat the washing step one more time.
    13. Prepare the 3% glutaraldehyde solution by adding 3 mL of glutaraldehyde to 97 mL of distilled water.
    14. Fix the co-cultured cells by adding 250 µL of 3% solution to the chamber wells and incubating for 1 h.
    15. Aspirate the fixative and wash the chamber wells as detailed from step 3.1.13.
    16. Dismantle the chamber wells using a tool that was provided with the chamber slides.
    17. Add a drop of the antifade compound, 1,4-diazabicyclo-[2.2.2]-octane to the slide to prevent auto-bleaching. Cover with a coverslip and seal the sides with a nail polish to prevent evaporation.
    18. View the co-cultured cells using the 100x objective lens (with oil) of a confocal laser-scanning microscope.
      NOTE: It is important to take pictures in bright-field and fluorescence to view interaction between amoeba and cryptococcal cells. Wherever possible, the fluorescence can be super-imposed onto the bright-field images. Amoeba cells are typically larger in size (i.e., 45-60 µm), and the trophozoite cells have an irregular shape. Cryptococcal cells are 5-10 µm in diameter and have a globose to ovoid shape. When exposed to a laser, it is possible that unstained amoeba cells may emit auto-fluorescence. Refer to Beisker and Dolbeare19 and Clancy and Cauller20 for methods to reduce autofluorescence.
  2. Acquiring quantitative data through use of fluorescence plate reader
    NOTE: Perform this assay with Acanthamoeba castellanii and C. neoformans UOFS Y-1378 or C. neoformans LMPE 046.
    1. Dispense a 100 µL suspension of standardized amoebae (adjusted to 1 x 107 cells/mL in ATCC medium 712) into a black, adherent 96 well microtiter plate.
    2. Incubate the plate for 2 h at 30 °C to allow amoeba cells to adhere to the surface.
    3. While amoeba cells are settling down to adhere, stain the standardized C. neoformans UOFS Y-1378 cells that were adjusted to 1 x 106 cells/mL (in 999 µL of PBS) with 1 µL of pHrodo Green Zymosan A BioParticles in a 1.5 mL microcentrifuge tube. Stain C. neoformans LMPE 046 cells as well in a separate tube.
      NOTE: The dye, unlike FITC, selectively stains cells that are trapped inside the acidic environment of a phagocytic cell21,22. For this technique, it is important to maintain the cryptococcal cells in a medium with a neutral pH (PBS) and amoeba in a medium with a neutral pH (ATCC medium 712). A medium with an acidic environment will result in a false positive reading of the relative fluorescence units, implying that a greater number of cryptococcal have been internalized.
    4. Gently agitate cryptococcal cells on an orbital shaker set at 50 rpm for 2 h at RT and in the dark.
    5. After 2 h, centrifuge the microcentrifuge tube at 960 x g for 5 min at 30 °C to pellet the cells. Aspirate the supernatant to remove PBS with the stain.
    6. Add 1 mL of PBS to the tube to wash the pelleted cells. Wash the cells by gentle pipetting.
    7. Centrifuge the cells at 960 x g for 5 min at 30 °C. Discard the supernatant. Repeat the washing step one more time.
    8. Resuspend the pellet of washed cells in 1 mL of PBS.
    9. Dispense a 100 µL suspension of stained cryptococcal cells to wells containing unstained amoeba cells.
    10. Incubate the prepared co-culture at 30 °C for an additional 2 h period.
      NOTE: The co-culture can be incubated for different timepoints to suit the purpose of the experiment.
    11. At the end of the co-incubation period, measure the fluorescence on a microplate reader. Convert logarithmic signals to relative fluorescence units.
      NOTE: The dye's excitation is at 492 nm and emission is at 538 nm. Consult Beisker and Dolbeare19 and Clancy and Cauller20 for methods to reduce autofluorescence.

4. Use of transmission electron microscopy to study phagocytosis (modified from van Wyk and Wingfield 23 )

  1. Add a 5 mL suspension of amoebae (adjusted to 1 x 107 cells/mL in ATCC medium 712) to a 15 mL centrifuge tube and allow them to settle for 30 min at 30 °C.
  2. Add a 5 mL suspension of C. neoformans UOFS Y-1378 cells (adjusted to 1 x 106 cells/mL in PBS) to the same centrifuge tube that contains 5 mL of standardized amoeba cells.
  3. Allow the tube stand for 2 h at 30 °C.
  4. Centrifuge the tube at 640 x g for 3 min at 30 °C to pellet the co-cultured cells. Aspirate the supernatant. Do not wash the co-cultured cells.
  5. Fix the co-cultured cells by resuspending the pellet in 3 mL of 1.0 M (pH = 7.0) sodium phosphate-buffered 3% glutaraldehyde for 3 h.
  6. Centrifuge the tube at 1,120 x g for 5 min at 30 °C to pellet the co-cultured cells. Aspirate the supernatant.
  7. Add 5 mL of sodium phosphate buffer to the centrifuge tube to wash the pelleted cells. Wash by gently pipetting the contents of the tube for 20 s.
  8. Centrifuge the tube at 1,120 x g for 5 min at 30 °C to pellet the co-cultured cells.
  9. Repeat steps 4.8-4.10. Aspirate the supernatant.
  10. Fix the co-cultured cells again by resuspending the pellet in 3 mL of 1.0 M (pH = 7.0) sodium phosphate-buffered 1% osmium tetroxide for 1.5 h.
  11. Remove the fixative (osmium tetroxide) by washing the co-cultured cells in a similar manner to removing 3% glutaraldehyde.
  12. Dehydrate the TEM material (also known as the co-cultured cells) in a graded acetoneseries of 30%, 50%, 70%, 95% and two changes of 100% for 15 min each, respectively. To do so, add 3 mL of the acetone solution to the pelleted cells and let it stand for 15 min. Then, centrifuge at 200 x g for 10 min at RT Discard the supernatant and add the higher percentage of the acetone solution.
  13. Prepare the epoxy of normal consistency according to the protocol by Spur24.
    NOTE: The epoxy resin is used for sectioning.
  14. Embed the TEM material into the freshly prepared epoxy resin. To do so, follow the steps below.
    1. Add 3 mL of the freshly prepared epoxy to a tube that contains the TEM material resuspended in 3 mL of 100% solution of acetone. Allow the tube to stand for 1 h.
    2. Centrifuge the tube at 200 x g for 10 min at 30 °C. Aspirate the epoxy-acetone solution.
    3. Add 6 mL of the freshly prepared epoxy to the pellet in the tube. Allow the tube to stand for 1 h.
    4. Centrifuge at 200 x g for 10 min at 30 °C. Aspirate all the epoxy-acetone solution.
    5. Add 3 mL of the freshly prepared epoxy to the tube. Allow the tube to stand for 8 h.
    6. Centrifuge at 200 x g for 10 min at 30 °C. Aspirate all the epoxy solution
    7. Add 3 mL of the freshly prepared epoxy to the tube. Keep the TEM material in the epoxy solution overnight in a vacuum desiccator.
      CAUTION: Epoxy resin is a radioactive material. Use PPE to handle the epoxy resin. The epoxy resin should also be handled in a fume hood. Researchers should follow safety regulations for discarding such material as specified by each country25.
  15. Polymerize the TEM material for 8 h at 70 °C.
  16. On the ultramicrotome, trim small sections of approximately 0.1 mm x 0.1 mm and 60 nm thickness from the epoxy-embedded material with a mounted glass knife. Assemble sections on a grid and place the grids in a TEM sample holder box before staining.
  17. Stain the sections with a drop of 6% uranyl acetate for 10 min in the dark. Ensure the sections are completely covered.|
    NOTE: Reconstitute the stain (6 g) in 100 mL of distilled water.
    CAUTION: Uranyl acetate is a radioactive material. Use PPE to handle uranyl acetate. Uranyl acetate should also be handled in a fume hood. Researchers should follow safety regulations for discarding such material as specified by each country25.
  18. Rinse sections by dipping them five times into a beaker that contains 100 mL of distilled water.
    NOTE: The distilled water should be disposed accordingly as it contains traces of uranyl acetate.
  19. Stain the section with a drop of lead citrate for 10 min in the dark. Ensure the sections are completely covered.
    NOTE: Lead citrate should be prepared according to the protocol by Reynold26.
  20. Rinse sections by dipping them five times into a beaker that contains 100 mL of distilled water.
  21. Individually assemble the grids with stained sections on a TEM sample holder box.
  22. View sections with a transmission electron microscope.

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Results

Microbes are microscopic organisms that cannot be perceived with the naked eye. However, their impact may result in observable clinically evident illnesses, such as skin infections. When studying certain aspects of microbes, ranging from their morphology, byproducts, and interactions, being able to provide pictorial and video evidence is of the utmost importance.

We first sought to visualize the interaction between cryptococcal ...

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Discussion

In the paper, different techniques were successfully employed to reveal the possible outcome that may arise when amoeba interact with cryptococcal cells. Also, we were interested to show the effects of 3-hydroxy fatty acids on the outcome of Cryptococcus-amoeba interactions.

The first technique used was confocal microscopy, which rendered still images. The major drawback of this technique here was that it only gave us information that is limited to a particular timepoint. Any conclusi...

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

The work was supported by a grant from the National Research Foundation of South Africa (grant number: UID 87903) and the University of the Free State. We are also grateful to services and assistance offered by Pieter van Wyk and Hanlie Grobler during our microscopy studies.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1,4-Diazabicyclo-[2.2.2]-octaneSigma-AldrichD27802-
1.5-mL plastic tube Thermo Fisher Scientific69715-
15-mL Centrifuge tube Thermo Fisher Scientific7252018-
50-mL Centrifuge tube Thermo Fisher Scientific1132017-
8-Well chamber slideThermo Fisher Scientific1109650-
AcetoneMerckSAAR1022040LC-
Amoeba strainATCCÒ30234TM-
ATCC medium 712ATCCÒ712TMAmoeba medium
Black 96-well microtiter plateThermo Fisher Scientific152089-
CentrifugeHermle--
ChloroformSigma-AldrichC2432-
Confocal microscopeNikonNikon TE 2000-
Epoxy resin:
[1] NSA[1] ALS[1] R1054-
[2] DER 736[2] ALS[2] R1073-
[3] ERL Y221 resin[3] ALS[3] R1047R-
[4] S1 (2-dimethylaminoethanol)[4] ALS [4] R1067-
Fluorescein isothiocyanateSigma-AldrichF4274-
Formic AcidSigma-Aldrich489441-
Fluoroskan Ascent FLThermo Fisher Scientific374-91038CMicroplate reader
GlucoseSigma-AldrichG8270-
GlutaraldehydeALSR1009-
HemocytometerBoeco--
Lead citrateALSR1209-
Liquid Chromatography Mass SpectrometerThermo Fisher Scientific-
MethanolSigma-AldrichR 34,860-
Orbital shakerLasec --
Osmium tetroxideALSR1015-
pHrodo Green Zymosan A BioParticlesLife TechnologiesP35365This is the pH-sensitive dye
Physiological buffer solutionSigma-AldrichP4417-50TAB-
Rotary shakerLabcon--
Sodium phosphate buffer:
[1] di-sodium hydrogen orthophosphate dihydrate [1] Merck[1] 106580-
[2] sodium di-hydrogen orthophosphate dihydrate[2] Merck [2] 106345
Transmission electron microscopePhilipsPhilips EM 100 -
Trypan blue Sigma-AldrichT8154-
UltramicrotomeLeicaEM UC7-
Uranyl acetateALSR1260A-
Vacuum dessicatorLasec --
VialSigma-Aldrich29651-U-
YNBLasec 239210-
YPD agarSigma-AldrichY-1500-

References

  1. Barton, L. L., Northup, D. E. Microbial Ecology. , Wiley-Blackwell. New Jersey. (2011).
  2. Hunter, P. Entente cordiale: multiple symbiosis illustrates the intricate interconnectivity of nature. EMBO Reports. 7, 861-864 (2006).
  3. Comolli, L. R. Intra- and inter-species interactions in microbial communities. Frontiers in Microbiology. , e629(2014).
  4. Siddiqui, R., Khan, N. A. Acanthamoeba is an evolutionary ancestor of macrophages: a myth or reality? Experimental Parasitology. 130, 95-97 (2012).
  5. Khan, N. A. Acanthamoeba: Biology and Pathogenesis. , Caister Academic Press. Nottingham. (2014).
  6. Steenbergen, J. N., Casadevall, A. The origin and maintenance of virulence for the human pathogenic fungus Cryptococcus neoformans. Microbes and Infection. 5, 667-675 (2003).
  7. Casadevall, A., Perfect, J. R. Cryptococcus Neoformans. , ASM Press. Washington D.C. (1998).
  8. Axelsson-Olsson, D., Olofsson, J., Ellstrom, P., Waldenstrom, J., Olsen, B. A simple method for long term storage of Acanthamoeba species. Parasitology Research. 104, 935-937 (2009).
  9. Sebolai, O. M., et al. 3-Hydroxy fatty acids found in capsules of Cryptococcus neoformans. Canadian Journal of Microbiology. 53, 809-812 (2007).
  10. Sebolai, O. M., Pohl, C. H., Botes, P. J., van Wyk, P. W. J., Kock, J. L. F. The influence of acetylsalicylic acid on oxylipin migration in Cryptococcusneoformans var. neoformans UOFS Y-1378. Canadian Journal of Microbiology. 54, 91-96 (2008).
  11. Yap, A. S., Michael, M., Parton, R. G. Seeing and believing: recent advances in imaging cell-cell interactions. F1000 Research. , e6435.1(2015).
  12. Follain, G., Mercier, L., Osmani, N., Harlepp, S., Goetz, J. G. Seeing is believing - multiscale spatio-temporal imaging towards in vivo cell biology. Journal of Cell Science. 130, 23-38 (2017).
  13. Lewis, L. E., Bain, J. M., Okai, B., Gow, N. A. R., Erwig, L. P. Live-cell video microscopy of fungal pathogen phagocytosis. Journal of Visualized Experiments. , e50196(2013).
  14. Duane, E. G. A practical guide to implementing a BLS-2+ biosafety program in a research laboratory. Applied Biosafety. 18, 30-36 (2013).
  15. Madu, U. L., et al. Cryptococcal 3-hydroxy fatty acids protect cells against amoebal phagocytosis. Frontiers in Microbiology. , e1351(2015).
  16. Smith, D., Onions, A. H. S. The Preservation and Maintenance of Living Fungi: IMI Technical Handbooks No. 2. , CAB International. Wallington. (1994).
  17. Schuster, F. L. Cultivation of pathogenic and opportunistic free-living amebas. Clinical Microbiology Reviews. 15, 342-344 (2002).
  18. Strober, W. Trypan blue exclusion test of cell viability. Current Protocols in Immunology. 21, 1-2 (2001).
  19. Beisker, W., Bolbeare, F., Gray, J. W. An improved immunocytochemical procedure for high sensitivity dectection of incorporated bromodeoxyuridine. Cytometry. 8, 235-239 (1987).
  20. Clancy, B., Cauller, L. J. Reduction of background autofluorescence in brain sections following immersion in sodium borohydride. Journal of Neuroscience Methods. 83, 97-102 (1998).
  21. Simons, E. R. Measurement of phagocytosis and of phagosomal environment in polymorphonuclear phagocytes by flow cytometry. Current Protocols in Cytometry. , Unit 9, 31 (2010).
  22. Grillo-Hill, B. K., Webb, B. A., Barber, D. L. Ratiometric Imaging of pH Probes. MethodsinCellBiology. 123, 429-448 (2014).
  23. van Wyk, P. W. J., Wingfield, M. J. Ascospores ultrastructure and development in Ophiostoma cucullatum. Mycologia. 83, 698-707 (1991).
  24. Spur, A. R. A low viscosity epoxy resin embedding medium for electron microscopy. Ultrastructural Research. 26, 31-43 (1969).
  25. Department of Minerals and Energy. Radioactive Waste Management Policy and Strategy for the Republic of South Africa. , Department of Minerals and Energy. Pretoria. (2005).
  26. Reynolds, E. S. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Cell Biology. 17, 208-212 (1963).
  27. Monici, M. Cell and tissue autofluorescence research and diagnostic applications. Biotechnology Annual Review. 11, 227-256 (2005).
  28. Janeway, C. A., Travers, P., Walport, M., Shlomchik, M. Immunobiology: The Immune System in Health and Disease, 5th edition. , Garland Science. New York. (2001).
  29. Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. Expulsion of live pathogenic yeast by macrophages. Current Biology. 16, 2156-2160 (2006).
  30. Alvarez, M., Casadevall, A. Phagosome extrusion and host-cell survival after Cryptococcusneoformans phagocytosis by macrophages. Current Biology. 16, 2161-2165 (2006).
  31. Madu, U. L., Ogundeji, A. O., Pohl, C. H., Albertyn, J., Sebolai, O. M. Elucidation of the role of 3-hydroxy fatty acids in Cryptococcus-amoeba interactions. Frontiers in Microbiology. , e765(2017).
  32. Hayes, B. K., Heit, E. Inductive reasoning 2.0. , Wiley Interdisciplinary Review: Cognitive Science. e1459(2018).
  33. Meindl, C., Öhlinger, K., Ober, J., Roblegg, E., Fröhlich, E. Comparison of fluorescence-based methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro. Toxicology. 378, 25-36 (2017).

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Confocal MicroscopyTransmission Electron MicroscopyPhagocytosis AssaypHrodo StainingFungal Cell CultureAmoeba ViabilityCo culture PreparationUltrastructural Analysis
Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions
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