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.

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 )
<ol>
	<li>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&#39;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.</li>
	<li>Incubate the agar plates for 48 h at 30 &#176;C. 
	NOTE: A plate can be stored for up to 2 months at 4 &#176;C before it can be discarded or used to make a stock culture16.</li>
	<li>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&#39;s ingredients can be found in Table 2.</li>
	<li>Incubate the flasks at 30 &#176;C for 24 h while agitating at 160 rpm on a rotary shaker.</li>
	<li>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.</li>
</ol>
2. Cultivation and standardization of amoeba cells (modified from Madu et al. 15 )
<ol>
	<li>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.</li>
	<li>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&#39;s ingredients can be found in Table 3.</li>
	<li>Manually shake it gently and immediately centrifuge for 5 min at 400 x g and 30 &#176;C.</li>
	<li>Aspirate the supernatant.</li>
	<li>Resuspend the cells in 15 mL of ATCC medium 712 and incubate the tube at 30 &#176;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.</li>
	<li>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.</li>
	<li>Incubate the tube at 30 &#176;C for 1 week while agitating at 160 rpm on a rotary shaker.</li>
	<li>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.</li>
	<li>Perform a viability assay using a trypan blue stain as detailed by Strober18. Proceed further with the cultures that show at least 80% viability.</li>
</ol>
3. Fluorescence staining of cells to study phagocytosis (modified from Madu et al. 15 )
<ol>
	<li>Gathering qualitative data through use of fluorescence microscope 
	NOTE: Perform this assay with Acanthamoeba castellanii and C. neoformans UOFS Y-1378.
	<ol>
		<li>Dispense a 200-&#181;L 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 &#176;C for cells to adhere to the surface.</li>
		<li>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 &#181;L of PBS) with 1 &#181;L 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.</li>
		<li>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.</li>
		<li>After 2 h, centrifuge at 960 x g for 5 min at 30 &#176;C to pellet the cells.</li>
		<li>Aspirate the supernatant to remove the PBS with the stain.</li>
		<li>Add 1 mL of PBS to the tube for washing the cell pellet. Wash the cells by gently pipetting.</li>
		<li>Centrifuge the cells at 960 x g for 5 min at 30 &#176;C. Discard the supernatant. Repeat the washing step one more time.</li>
		<li>Resuspend the washed cells in 1 mL of PBS.</li>
		<li>Dispense a 200 &#181;L suspension of the stained C. neoformans UOFS Y-1378 cells to chamber wells containing the unstained amoeba cells.</li>
		<li>Incubate the prepared co-culture at 30 &#176;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.</li>
		<li>At the end of the co-incubation period, aspirate the contents of the wells.</li>
		<li>Add 300 &#181;L 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.</li>
		<li>Prepare the 3% glutaraldehyde solution by adding 3 mL of glutaraldehyde to 97 mL of distilled water.</li>
		<li>Fix the co-cultured cells by adding 250 &#181;L of 3% solution to the chamber wells and incubating for 1 h.</li>
		<li>Aspirate the fixative and wash the chamber wells as detailed from step 3.1.13.</li>
		<li>Dismantle the chamber wells using a tool that was provided with the chamber slides.</li>
		<li>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.</li>
		<li>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 &#181;m), and the trophozoite cells have an irregular shape. Cryptococcal cells are 5-10 &#181;m 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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Dispense a 100 &#181;L suspension of standardized amoebae (adjusted to 1 x 107 cells/mL in ATCC medium 712) into a black, adherent 96 well microtiter plate.</li>
		<li>Incubate the plate for 2 h at 30 &#176;C to allow amoeba cells to adhere to the surface.</li>
		<li>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 &#181;L of PBS) with 1 &#181;L 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.</li>
		<li>Gently agitate cryptococcal cells on an orbital shaker set at 50 rpm for 2 h at RT and in the dark.</li>
		<li>After 2 h, centrifuge the microcentrifuge tube at 960 x g for 5 min at 30 &#176;C to pellet the cells. Aspirate the supernatant to remove PBS with the stain.</li>
		<li>Add 1 mL of PBS to the tube to wash the pelleted cells. Wash the cells by gentle pipetting.</li>
		<li>Centrifuge the cells at 960 x g for 5 min at 30 &#176;C. Discard the supernatant. Repeat the washing step one more time.</li>
		<li>Resuspend the pellet of washed cells in 1 mL of PBS.</li>
		<li>Dispense a 100 &#181;L suspension of stained cryptococcal cells to wells containing unstained amoeba cells.</li>
		<li>Incubate the prepared co-culture at 30 &#176;C for an additional 2 h period. 
		NOTE: The co-culture can be incubated for different timepoints to suit the purpose of the experiment.</li>
		<li>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&#39;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.</li>
	</ol>
	</li>
</ol>
4. Use of transmission electron microscopy to study phagocytosis (modified from van Wyk and Wingfield 23 )
<ol>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>Allow the tube stand for 2 h at 30 &#176;C.</li>
	<li>Centrifuge the tube at 640 x g for 3 min at 30 &#176;C to pellet the co-cultured cells. Aspirate the supernatant. Do not wash the co-cultured cells.</li>
	<li>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.</li>
	<li>Centrifuge the tube at 1,120 x g for 5 min at 30 &#176;C to pellet the co-cultured cells. Aspirate the supernatant.</li>
	<li>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.</li>
	<li>Centrifuge the tube at 1,120 x g for 5 min at 30 &#176;C to pellet the co-cultured cells.</li>
	<li>Repeat steps 4.8-4.10. Aspirate the supernatant.</li>
	<li>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.</li>
	<li>Remove the fixative (osmium tetroxide) by washing the co-cultured cells in a similar manner to removing 3% glutaraldehyde.</li>
	<li>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.</li>
	<li>Prepare the epoxy of normal consistency according to the protocol by Spur24. 
	NOTE: The epoxy resin is used for sectioning.</li>
	<li>Embed the TEM material into the freshly prepared epoxy resin. To do so, follow the steps below.
	<ol>
		<li>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.</li>
		<li>Centrifuge the tube at 200 x g for 10 min at 30 &#176;C. Aspirate the epoxy-acetone solution.</li>
		<li>Add 6 mL of the freshly prepared epoxy to the pellet in the tube. Allow the tube to stand for 1 h.</li>
		<li>Centrifuge at 200 x g for 10 min at 30 &#176;C. Aspirate all the epoxy-acetone solution.</li>
		<li>Add 3 mL of the freshly prepared epoxy to the tube. Allow the tube to stand for 8 h.</li>
		<li>Centrifuge at 200 x g for 10 min at 30 &#176;C. Aspirate all the epoxy solution</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Polymerize the TEM material for 8 h at 70 &#176;C.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Rinse sections by dipping them five times into a beaker that contains 100 mL of distilled water.</li>
	<li>Individually assemble the grids with stained sections on a TEM sample holder box.</li>
	<li>View sections with a transmission electron microscope.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+the+role+of+3-hydroxy+fatty+acids+in+Cryptococcus-amoeba+interactions.">Elucidation of the role of 3-hydroxy fatty acids in Cryptococcus-amoeba interactions.</a> Frontiers in Microbiology. , e765 (2017).</li><li>Hayes, B. K., Heit, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inductive reasoning 2.0. , e1459 (2018).</li><li>Meindl, C., &#214;hlinger, K., Ober, J., Roblegg, E., Fr&#246;hlich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fluorescence-based+methods+to+determine+nanoparticle+uptake+by+phagocytes+and+non-phagocytic+cells+in+vitro.">Comparison of fluorescence-based methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro.</a> Toxicology. 378, 25-36 (2017).</li></ol>

The authors declare that they have no competing financial interests.

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 conclusion that can be drawn based on the results lends itself to inductive reasoning, wherein one can arrive at a conclusion based on a set of observations32. However, just because one observes several situations in which a pattern exists does not mean that that pattern is true for all situations. Thus, in the study, it is shown and possibly cautioned how such limited information may lead to unfounded conclusions. To the point, in the absence of contradictory or supportive, complementary evidence, it may be concluded that internalization may have led to the phagocytosis of cells.
The pace of development in imaging brings new opportunities to make scientific discoveries, as was the case with the uncovering of vomocytosis29,30. To illustrate this point without use of a microscope that can record time-lapse videos, this discovery would have not been possible. Therefore, a lack of access to such high-end instrumentation will always be an obstacle in resource poor-settings that are not at the forefront of uncovering such processes. One way to overcome this is to seek out new collaborations or discover innovative ways to address research questions. One welcome development has been the introduction and application of specialized stains such as the phagocytic stain used here21,22. This stain is pH-sensitive and fluoresces only in acid environments such as in the lumen of amoeba food vacuole15. It is worthwhile to point out that the stain only gives information related to the internalization of cells. Determination if cells are eventually phagocytized in additional experiments may be required.
Importantly, such a stain also proved to be useful in the measurement of fluorescence. The latter allowed integration of quantitative data in an attempt to explain what happens biologically at one specific timepoint. Here, fate of cells was discerned (i.e., it was determined whether the presence of 3-hydroxy fatty acids impaired or promoted the internalization of cells) by extrapolating meaning from the readings of relative fluorescence units.
Unlike in this study, researchers may also opt to measure the fluorescence of cells over a time period. The obtained information is useful in determining the number of cells that are internalized at one timepoint and following how the amount changes over the period. Likewise, images can also be taken at corresponding timepoints.
This study shows the power of combining a number of methods to reach a reasoned conclusion. The approach of combining multiple approaches to monitor phagocytosis either to compare or complement an initial technique is not new. For example, Meindl and co-workers33 compared three techniques (image analysis, fluorescence, and flow cytometry readings) to investigate how fluorescence-labelled particle size affects macrophage phagocytosis. The study proved that of the three techniques, plate reading may be the best option to monitor phagocytosis33.
TEM is particularly a powerful tool, as it provides a bird&#39;s eye view into the lumen of the food vacuole. Often, this level of detail is frequently missed by confocal microscopy in the form of still images, including time-lapse videos. To this point of the TEM, it was interesting to visualize protuberances on the surfaces of the cryptococcal capsule. It was previously hypothesized that these cell surface structures are used as a channel to release 3-hydroxy fatty acids into the surrounding environment to possibly promote cell survival9,10,15,31. The detail on the TEM micrograph further reveals that protuberances on the internalized cell are not distorted and have maintained their integrity. Thus (given the integrity of the protuberances), it is possible that they may deliver 3-hydroxy fatty acids into the food vacuole environment and alter internal conditions, leading to cell survival as reported by Madu et al.15,31. A major limitation of using the electron microscope is that sample preparation is very laborious. Moreover, to avert destroying the samples as seen in Figure 4, the experimenter should be well-trained to manually operate the ultramicrotome and microscope.
In conclusion, it is envisaged that researchers will be encouraged by the prospect of studying phagocytosis simply by combining still fluorescent images with quantitative data. It is trusted that researchers can obtain enough information from this protocol and optimize it in their own studies. This may include the development of antibodies against targeted metabolites and applying this to immunofluorescence studies, including immuno-gold labelling during TEM examination.

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.

<table><tbody><tr><td>1,4-Diazabicyclo-[2.2.2]-octane</td><td>Sigma-Aldrich</td><td>D27802</td><td>-</td></tr><tr><td>1.5-mL plastic tube&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>69715</td><td>-</td></tr><tr><td>15-mL Centrifuge tube&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>7252018</td><td>-</td></tr><tr><td>50-mL Centrifuge tube&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>1132017</td><td>-</td></tr><tr><td>8-Well chamber slide</td><td>Thermo Fisher Scientific</td><td>1109650</td><td>-</td></tr><tr><td>Acetone</td><td>Merck</td><td>SAAR1022040LC</td><td>-</td></tr><tr><td>Amoeba strain</td><td>ATCC&lt;sup&gt;&amp;Ograve;&lt;/sup&gt;</td><td>30234&lt;sup&gt;TM&lt;/sup&gt;</td><td>-</td></tr><tr><td>ATCC medium 712</td><td>ATCC&lt;sup&gt;&amp;Ograve;&lt;/sup&gt;</td><td>712&lt;sup&gt;TM&lt;/sup&gt;</td><td>Amoeba medium</td></tr><tr><td>Black 96-well microtiter plate</td><td>Thermo Fisher Scientific</td><td>152089</td><td>-</td></tr><tr><td>Centrifuge</td><td>Hermle</td><td>-</td><td>-</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>C2432</td><td>-</td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>Nikon TE 2000</td><td>-</td></tr><tr><td>Epoxy resin:</td><td></td><td></td><td></td></tr><tr><td>[1] NSA</td><td>[1] ALS</td><td>[1] R1054</td><td>-</td></tr><tr><td>[2] DER 736</td><td>[2] ALS</td><td>[2] R1073</td><td>-</td></tr><tr><td>[3] ERL Y221 resin</td><td>[3] ALS</td><td>[3] R1047R</td><td>-</td></tr><tr><td>[4] S1 (2-dimethylaminoethanol)</td><td>[4] ALS&amp;nbsp;</td><td>[4] R1067</td><td>-</td></tr><tr><td>Fluorescein isothiocyanate</td><td>Sigma-Aldrich</td><td>F4274</td><td>-</td></tr><tr><td>Formic Acid</td><td>Sigma-Aldrich</td><td>489441</td><td>-</td></tr><tr><td>Fluoroskan Ascent FL</td><td>Thermo Fisher Scientific</td><td>374-91038C</td><td>Microplate reader</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td>-</td></tr><tr><td>Glutaraldehyde</td><td>ALS</td><td>R1009</td><td>-</td></tr><tr><td>Hemocytometer</td><td>Boeco</td><td>-</td><td>-</td></tr><tr><td>Lead citrate</td><td>ALS</td><td>R1209</td><td>-</td></tr><tr><td>Liquid Chromatography Mass Spectrometer</td><td>Thermo Fisher Scientific</td><td></td><td>-</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>R 34,860</td><td>-</td></tr><tr><td>Orbital shaker</td><td>Lasec&amp;nbsp;</td><td>-</td><td>-</td></tr><tr><td>Osmium tetroxide</td><td>ALS</td><td>R1015</td><td>-</td></tr><tr><td>pHrodo Green Zymosan A BioParticles</td><td>Life Technologies</td><td>P35365</td><td>This is the pH-sensitive dye</td></tr><tr><td>Physiological buffer solution</td><td>Sigma-Aldrich</td><td>P4417-50TAB</td><td>-</td></tr><tr><td>Rotary shaker</td><td>Labcon</td><td>-</td><td>-</td></tr><tr><td>Sodium phosphate buffer:</td><td></td><td></td><td></td></tr><tr><td>[1] di-sodium hydrogen orthophosphate dihydrate&amp;nbsp;</td><td>[1] Merck</td><td>[1] 106580</td><td>-</td></tr><tr><td>[2] sodium di-hydrogen orthophosphate dihydrate</td><td>[2] Merck&amp;nbsp;</td><td>[2] 106345</td><td></td></tr><tr><td>Transmission electron microscope</td><td>Philips</td><td>Philips EM 100&amp;nbsp;</td><td>-</td></tr><tr><td>Trypan blue&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T8154</td><td>-</td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td>EM UC7</td><td>-</td></tr><tr><td>Uranyl acetate</td><td>ALS</td><td>R1260A</td><td>-</td></tr><tr><td>Vacuum dessicator</td><td>Lasec&amp;nbsp;</td><td>-</td><td>-</td></tr><tr><td>Vial</td><td>Sigma-Aldrich</td><td>29651-U</td><td>-</td></tr><tr><td>YNB</td><td>Lasec&amp;nbsp;</td><td>239210</td><td>-</td></tr><tr><td>YPD agar</td><td>Sigma-Aldrich</td><td>Y-1500</td><td>-</td></tr></tbody></table>

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.

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 cells and amoeba. For this purpose, bright-field images that showed 2 h co-incubated cells were studied first. One image revealed a cryptococcal cell that was in the close proximity to amoeba. One of the amoeba cells was seen with extended pseudopodia to capture a cryptococcal cell (Figure 1A). Next, a corresponding image in fluorescence was captured for referencing (Figure 1B). The green fluorescence on the surface of the stained cells aided in confirming the presence of cryptococcal cells. The unstained amoeba also auto-fluoresced. This, in addition to the apparent difference size and morphology, assisted in further distinguishing the two cell types.
Autofluorescence is a quality often observed when biological structures naturally emit light that they have absorbed (e.g., following exposure to a laser during confocal laser scanning microscopy)27. In Figure 1C, cryptococcal cells were noted (at the same timepoint of 2 h) that were already internalized by amoeba. The corresponding image in fluorescence was also captured for referencing (Figure 1D). Based on the evidence at hand, it is tempting to conclude that the amoeba killed the two trapped cells. However, phagocytosis is a dynamic process wherein the host, predator and pathogen, and prey employ different strategies to destroy or evade each other28. The act of cryptococcal cells evading phagocytic cells is elegantly demonstrated by vomocytosis29,30, which is a non-lytic expulsion process of trapped cells from macrophages. This daring move has been captured in time-lapse videos29,30. Unfortunately, this highlights the limitation of studying still images of fixed cells, as in our study, to elucidate a dynamic process like phagocytosis. To the point, a researcher may miss the interval when a cell escapes from its capturer.
To compensate for the above, the reading of relative fluorescence units was considered. In the current study, readings were taken after a 2 h co-incubation period and helped to compare the response of the two test cryptococcal strains [i.e., one that produces 3-hydroxy fatty acids (C. neoformans UOFS Y-1378) and the other that does not (C. neoformans LMPE 046)]. It was hypothesized that 3-hydroxy fatty acids may act as a virulence determinant that impair the uptake of cryptococcal cells, including phagocytosis by amoeba. For more information on the influence of 3-hydroxy fatty acids on amoeba, it is advised to refer to Madu et al.15,31. Figure 2 shows the amount of cryptococcal cells that were internalized based on the reading of fluorescence units. When comparing the two cryptococcal isolates, it was clear that cells that produce the 3-hydroxy fatty acids were internalized less frequently compared to cells that do not produce 3-hydroxy fatty acids.
To enhance the qualitative data, transmission electron microscopy was included in the analysis (Figure 3A). Here, it was noted that the strain that produces 3-hydroxy fatty acids (C. neoformans UOFS Y-1378) had spiky protuberances on the capsule (Figure 3B), which may be used by the cell to release 3-hydroxy fatty acids to the outside environment.
It is important to note that the data (in Figure 1, Figure 3) convey the fate of cryptococcal cells as being internalized and not killed/phagocytized. To determine if the cells survived the phagocytic event, it is recommended to include an additional assay in which the researcher lyses the amoeba cells and prepares a spread plate agar to enumerate the cryptococcal colony forming units (CFU). By counting CFUs, Madu et al.15 reported that cryptococcal cells producing 3-hydroxy fatty acids were also resistant to the phagocytic action of amoeba following internalization. Thus, these cells yielded a significantly higher survival rate when compared to cells that do not produce 3-hydroxy fatty acids.
Figure&#160;4 shows the importance of TEM sample preparation and examination. In this instance, C. neoformans UOFS Y-1378 sections were purposefully overexposed to electron bombardment. At the end, the captured image cannot be used, as it compromises the quality of information that can be deduced. Taken together, the obtained information shows that by combining these different techniques, a researcher is able to deduce sufficient information to determine the fate of cryptococcal cells when co-cultured with amoeba.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>Quantity</td>
		</tr>
		<tr>
			<td>bacteriological peptone</td>
			<td>20 g/L</td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>10 g/L</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>20 g/L</td>
		</tr>
		<tr>
			<td>agar</td>
			<td>15 g/L</td>
		</tr>
	</tbody>
</table>
Table 1: Ingredients for making YPD agar. Add the required amount all the ingredients in 1 L of water. Heat while stirring to dissolve the ingredients completely. Once done autoclave prior to the use.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>Quantity</td>
		</tr>
		<tr>
			<td>ammonium sulfate&#160;</td>
			<td>5 g/L</td>
		</tr>
		<tr>
			<td>biotin</td>
			<td>2 &#956;g/L</td>
		</tr>
		<tr>
			<td>calcium pantothenate</td>
			<td>400 &#956;g/L</td>
		</tr>
		<tr>
			<td>folic acid&#160;</td>
			<td>2 &#956;g/L</td>
		</tr>
		<tr>
			<td>inositol</td>
			<td>2000 &#956;g/L</td>
		</tr>
		<tr>
			<td>niacin&#160;</td>
			<td>400 &#956;g/L</td>
		</tr>
		<tr>
			<td>p-aminobenzoic acid</td>
			<td>200 &#956;g/L</td>
		</tr>
		<tr>
			<td>pyridoxine hydrochloride</td>
			<td>400 &#956;g/L</td>
		</tr>
		<tr>
			<td>riboflavin&#160;</td>
			<td>200 &#956;g/L</td>
		</tr>
		<tr>
			<td>thiamine hydrochloride</td>
			<td>400 &#956;g/L</td>
		</tr>
		<tr>
			<td>boric acid</td>
			<td>500 &#956;g/L</td>
		</tr>
		<tr>
			<td>copper sulfate</td>
			<td>&#160;40 &#956;g/L</td>
		</tr>
		<tr>
			<td>potassium iodide</td>
			<td>100 &#956;g/L</td>
		</tr>
		<tr>
			<td>ferric chloride</td>
			<td>200 &#956;g/L</td>
		</tr>
		<tr>
			<td>manganese sulfate</td>
			<td>400 &#956;g/L</td>
		</tr>
		<tr>
			<td>sodium molybdate&#160;</td>
			<td>200 &#956;g/L</td>
		</tr>
		<tr>
			<td>zinc sulfate&#160;</td>
			<td>400 &#956;g/L</td>
		</tr>
		<tr>
			<td>monopotassium phosphate</td>
			<td>&#160;1 g/L</td>
		</tr>
		<tr>
			<td>magnesium sulfate</td>
			<td>&#160;0.5 g/L</td>
		</tr>
		<tr>
			<td>sodium chloride&#160;</td>
			<td>0.1 g/L</td>
		</tr>
		<tr>
			<td>calcium chloride&#160;</td>
			<td>0.1 g/L</td>
		</tr>
	</tbody>
</table>
Table 2: Ingredients for making YNB broth. Add the required amount all the ingredients in 1 L of water. Heat while stirring to dissolve the ingredients completely. Once done autoclave prior to the use.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Part I: Basal medium.&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ingredient</td>
			<td>Quantity</td>
		</tr>
		<tr>
			<td>proteose peptone&#160;</td>
			<td>20 g/L</td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>1 g/L</td>
		</tr>
		<tr>
			<td>agar (if needed)</td>
			<td>20 g/L</td>
		</tr>
		<tr>
			<td>Part II: Supplements.&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ingredient (stock solutions)</td>
			<td>Quantity</td>
		</tr>
		<tr>
			<td>0.05 M CaCl2</td>
			<td>8 mL</td>
		</tr>
		<tr>
			<td>0.4 M MgSO4 x 7H2O</td>
			<td>10 ml&#160;</td>
		</tr>
		<tr>
			<td>0.25 M Na2HPO4 x 7H2O</td>
			<td>10 mlL</td>
		</tr>
		<tr>
			<td>0.25 M KH2PO4</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Na Citrate x 2H2O</td>
			<td>1 g&#160;</td>
		</tr>
		<tr>
			<td>0.005 M Fe(NH4)2(SO4)2 x 6H2O&#160;</td>
			<td>10 mL</td>
		</tr>
	</tbody>
</table>
Table 3: Ingredients for making ATCC medium 712. Prepare the basal medium in 900 mL of water. Prepare the supplements separately and add to the basal medium. Once done adjust the pH to 7.4 with 1 N HCl or 1 N NaOH and autoclave. Filter sterilize 50 mL solution of 2 M glucose (18 g/50 mL) and add it aseptically to the complete medium prior to use.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58698/58698fig1.jpg" /> 
Figure 1: Bright-field and corresponding fluorescent micrographs showing amoeba-Cryptococcus interactive moments. (A) An amoeba cell in close proximity to a C. neoformans UOFS Y-1378 cell can be seen. The corresponding fluorescent image is shown in (B). (C) Depiction of two C. neoformans UOFS Y-1378 cells that are trapped inside the amoeba food vacuole. The corresponding fluorescent image is shown in (D). This figure has been modified from Madu et al.15. A = amoeba; C = C. neoformans. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58698/58698fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58698/58698fig2.jpg" /> 
Figure 2: The results of the internalization assay of cryptococcal cells co-cultured with amoeba. The reading of relative fluorescence units allows for the interpretation and comparison of the efficiency of amoebae to internalize C. neoformans UOFS Y-1378 and C. neoformans LMPE 046. The error bars represent the calculated standard errors based on three biological replicates. This figure has been modified from Madu et al.15. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58698/58698fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58698/58698fig3.jpg" /> 
Figure 3: Transmission electron micrographs showing amoeba-Cryptococcus interactions. TEM micrographs (A, B) confirm the observations in Figure 1C,D. (A) Shown is a C. neoformans UOFS Y-1378 cell trapped inside the amoeba food vacuole, while (B) is a close-up view of Figure 3A.This figure has been modified from Madu et al.15. A = amoeba cell; C = C. neoformans cell. The red arrow points at a capsular protuberance. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58698/58698fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58698/58698fig4.jpg" /> 
Figure 4: A transmission electron micrograph showing C. neoformans UOFS Y-1378 cells. The cells are damaged and thus cannot provide meaningful data. Red arrows indicate points where the section is torn. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58698/58698fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1.&#160; <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58698/Supplemental_File_1.docx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions at JoVE.com

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.

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 ...

complementary-use-microscopic-techniques-fluorescence-reading

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

6.2K Views.  University of the Free State. 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.

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

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Bioengineering

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

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 ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

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 ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Biology

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

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

Summary

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Chapters in this video

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

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

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions