We would like to thank Jonathan Cooper Lab at Fred Hutchinson Cancer Research Center for the permission to use their cryostat. This work was supported by NIH and the W.M. Keck Foundation. BM is a Gordon and Betty Moore fellow of Life Science Research Foundation. We are grateful to Daniel Gottschling for sharing his fixing protocol with us.

1. Fixing Yeast Communities
<ol>
	<li>Grow yeast communities on a membrane filter (e.g., Millipore MF membrane filter; Figure 2A). This protocol corresponds to a typical community of less than 2x108 cells on a 6 mm-diameter membrane filter.</li>
	<li>Use a piece of Whatman 541 filter paper to cover the bottom of a 1 cm-diameter well (Figure 2B). This Whatman paper will be used as a carrier to transfer the community in later stages. Add 1 ml 100% methanol to the well, and leave the well in -20 &deg;C for the temperature to equilibrate.</li>
	<li>Put liquid N2 in a dewar. Freeze community in liquid nitrogen by dipping the community for at least 15 sec in liquid N2 using a tweezer (Figure 2C).</li>
	<li>Transfer frozen community to the well of -20 &deg;C methanol (Figure 2D). Ensure that the community is kept horizontal when submerging it in the methanol. Wait 20 min. The MF membrane filter starts to disintegrate in methanol.</li>
	<li>Transfer community using the carrier Whatman 541 filter paper from the methanol well to a cold Petri dish kept at -20 &deg;C for methanol evaporation (Figure 2E). When transferring, keep the community on top of carrier Whatman filter and draw away extra drops of methanol using another piece of thick Whatman paper before putting in the cold Petri dish. This will ensure that evaporation time is consistent from sample to sample.</li>
	<li>Wait 4-6 hr for evaporation until the carrier Whatman filter looks dry. Insufficient evaporation leaves residual methanol which interferes with sectioning. Over-drying causes cracks and deformation in communities.</li>
	<li>Take the sample out of -20 &deg;C freezer. Cut away Whatman 541 filter paper not covered by community.</li>
	<li>Make a rectangular cubic aluminum-foil container which is large enough such that the sample has at least ~2 mm margin from each side (Figure 2F). Place the community on the bottom surface of the aluminum-foil container; immediately cover the sample with OCT to 3~4 mm above community height. If important, the orientation of the sample can be adjusted in this step with regard to the sides of the container. Wait 10 min.</li>
	<li>Place the OCT-embedded sample on a metallic plate on dry ice to freeze. Store the frozen communities at -20 &deg;C or -80 &deg;C freezer.</li>
</ol>
2. Sectioning Frozen Communities for Fluorescence Imaging
<ol>
	<li>Set the temperature of cryosection chamber to -25 &deg;C and leave the samples in the chamber until its temperature reaches the temperature of the chamber.</li>
	<li>Mount the sample onto a cryo-mount after applying a drop of OCT on the mount. Spread OCT uniformly on the side of sample with exposed Whatman filter paper (bottom of the container) so that OCT thickness is 2-3 mm. Let OCT freeze inside the cryostat chamber before sectioning. Ensuring an OCT margin of 2-3 mm on all sides of the sample helps to preserve the integrity of sections.</li>
	<li>The sides of the OCT sample block can be used to align the sample. If the sample is not aligned with respect to the blade, sectioning will start from a corner or side of the sample. Adjust the mount to ensure that the blade cuts a perfectly vertical (or horizontal) face of the sample block.</li>
	<li>Typical sections for yeast communities are ~14 &mu;m-thick. After cutting a section, use a microscope slide kept at room temperature to collect the section, by slowly approaching the slide to the cut section. The section will melt and transfer to the microscope slide. Up to 18 sections can be transferred onto one slide.</li>
	<li>Store the slides at cold temperatures, preferably -20 &deg;C, before imaging. Loss of signal is minimal when kept for one week at 4 &deg;C. For maximum fluorescence signal, imaging is preferably done immediately after sectioning.</li>
</ol>

<ol>
	<li>Tolker-Nielsen, T., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Organization+of+Microbial+Biofilm+Communities.">Spatial Organization of Microbial Biofilm Communities.</a> Microbial Ecology. 40 (2), 75-84 (2000).</li><li>Moller, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+In+Situ+Gene+Expression+in+Mixed-Culture+Biofilms:+Evidence+of+Metabolic+Interactions+between+Community+Members."> In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members.</a> Appl. Environ. Microbiol. 64 (2), 721-732 (1998).</li><li>Lepp, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methanogenic+Archaea+and+human+periodontal+disease.">Methanogenic Archaea and human periodontal disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (16), 6176-6181 (2004).</li><li>V&#225;chov&#225;, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+developing+multicellular+yeast+colony:+spatio-temporal+expression+of+Ato1p+ammonium+exporter.">Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter.</a> Environmental Microbiology. 11 (7), 1866-1877 (2009).</li><li>Min&#225;rikov&#225;, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+Gene+Expression+in+Cells+within+Yeast+Colonies.">Differentiated Gene Expression in Cells within Yeast Colonies.</a> Experimental Cell Research. 271 (2), 296-304 (2001).</li><li>Shou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+cooperation+in+engineered+yeast+populations.">Synthetic cooperation in engineered yeast populations.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 1877-1882 (2007).</li><li>Momeni, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+generates+a+unique+spatial+signature+in+microbial+communities.">Cooperation generates a unique spatial signature in microbial communities.</a> , (2012).</li><li>Kiernan, J., ed, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histological and Histochemical Methods: Theory and Practice. , 4 (2008).</li><li>Piccirillo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rim101p/PacC+Pathway+and+Alkaline+pH+Regulate+Pattern+Formation+in+Yeast+Colonies.">The Rim101p/PacC Pathway and Alkaline pH Regulate Pattern Formation in Yeast Colonies.</a> Genetics. 184 (3), 707-716 (2010).</li></ol>

No conflicts of interest declared.

The procedure presented here offers a way to inspect the internal structure of yeast communities. The methanol fixing step makes the yeast colony rigid8 and preserves the integrity of the colony; however, it also causes shrinkage8 that should be taken into account in interpreting the results. We typically see around 30% shrinkage in height of yeast colonies, compared to colonies directly embedded in OCT without fixing9.
To avoid shrinkage, an alternative approach for cryosectioning is to directly embed the communities in OCT and freeze them. Fluorescence is preserved in the method based on direct embedding, but the drawback is that the cells within each section are not bound together. As a result, some of the cells at the edges of the section float away, and the sections are more likely to suffer disturbances in the sectioning process (Figure 4). Although not focused on here, bright-field images of cross-sections of yeast communities may also give us useful information about the state of cells within the community9. Whether or not the property of interest is affected by the fixing step has to be examined on a case-by-case basis.

<table><tbody><tr><td>100% Methanol</td><td>J.T. Baker</td><td>9070-01</td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Andwin Scientific</td><td>4583</td><td></td></tr><tr><td>Whatman Grade No. 541 Quantitative Filter Paper</td><td>Whatman</td><td>1541-047</td><td></td></tr><tr><td>Millipore-MF Membrane Filter</td><td>Millipore</td><td>HAWP04700</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM 1510 S</td><td></td></tr></tbody></table>

cryosectioning yeast communities for examining fluorescence patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community1. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells4. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells4.
Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.
Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

Fluorescence images of vertical cross-sections of yeast communities containing red- and green-tagged competitive populations6 are shown in Figure 3. These communities consist of two prototrophic strains of yeast and their competition for shared resources, including space, leads to columnar patterns7, as displayed in their vertical cross-sections. Resolutions down to a single cell can be resolved in cross-sections. Figure 3 compares cross-sections of replicate communities obtained with (bottom) and without (top) methanol-fixing, following the protocols in Figure 1. For cryosectioning yeast communities without fixing, the protocol is followed starting from step 1.8. The fluorescence signal is preserved in community sections without fixing, but the integrity of the community is compromised. As a result: (1) some cells float away around the edge of the section, and (2) the overall yield (i.e., fraction of sections not perturbed by artifacts of the sectioning procedure) is lower compared to fixed communities.
Figure 4 shows representative bright-field and fluorescence vertical cross-sections of a yeast community. Cells on top of the community form a crown that appears distinct from other cells characterized by stronger scattering in bright-field and brighter fluorescence. These patterns are consistent with reported patterns of differentiation in yeast colonies4.
<img src="/files/ftp_upload/50101/50101fig1.jpg" alt="Figure 1"/> 
 Figure 1. Flowchart of the typical procedure for cryosectioning yeast communities with (left) and without (right) fixing.
<img src="/files/ftp_upload/50101/50101fig2.jpg" alt="Figure 2"/> 
 Figure 2. Steps involved in the fixing and freezing of yeast communities are illustrated. (A) Yeast communities are grown on top of 6 mm-diameter disks of MF membrane filter. (B) A piece of Whatman 541 filter paper is put in a well containing methanol to be used as the carrier. (C) Communities are frozen by dipping them in liquid nitrogen for typically ~15 sec. (D) Frozen communities are transferred to -20 &deg;C well of methanol on top of carrier Whatman filter. (E) After fixing, the carrier Whatman filter is used to take communities out of methanol and transfer them to a cold dish for evaporation. (F) The community after evaporation is transferred to a rectangular cubic aluminum-foil container and covered with OCT for final freezing of the block.
<img src="/files/ftp_upload/50101/50101fig3.jpg" alt="Figure 3"/> 
 Figure 3. Representative fluorescence images of vertical cross-section of replicate yeast communities grown under identical conditions are shown without (top) and with (bottom) methanol-fixing. The community consists of two prototrophic strains of S. cerevisiae tagged with mCherry and yEGFP fluorescent proteins7. Without fixing, cells are not bound together and may float away or rearrange during sectioning. Methanol-fixing keeps community cells together, but causes shrinkage as observed in the difference between community heights (% average).
<img src="/files/ftp_upload/50101/50101fig4.jpg" alt="Figure 4"/> 
 Figure 4. Representative bright-field (top) and fluorescence (bottom) images of vertical cross-section of a yeast community are shown. The community consists of two prototrophic strains of S. cerevisiae tagged with dsRed and YFP fluorescent proteins7.

Watch this Scientific Journal Video about Cryosectioning Yeast Communities for Examining Fluorescence Patterns at JoVE.com

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

cryosectioning-yeast-communities-for-examining-fluorescence-patterns

Nanyang Technological University

Research

JoVE Journal

Biology

8.8K Views.  Fred Hutchinson Cancer Research Center. We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

Video: Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Biochemistry

Visualizing Yeast Organelles with Fluorescent Protein Markers

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

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Bioengineering

Protocol for Co-Culturing Sucrose-Secreting Cyanobacteria with Fungal Partners

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being explored as a method for solar-driven bioproduction. One approach involves the use of CO2-fixing cyanobacteria that secrete organic carbon to support the metabolism of a co-cultivated heterotroph, which in turn transforms the carbon into higher-value goods or services. In this protocol, a technical description to assist the experimentalist in the establishment of a co-culture combining a sucrose-secreting cyanobacterial strain with a fungal partner(s), as represented by model yeast species, is provided. The protocol describes the key prerequisites for co-culture establishment: Defining the media composition, monitoring the growth characteristics of individual partners, and the analysis of mixed cultures with multiple species combined in the same growth vessel. Basic laboratory techniques for co-culture monitoring, including microscopy, cell counter, and single-cell flow cytometry, are summarized, and examples of nonproprietary software to use for data analysis of raw flow cytometry standard (FCS) files in line with FAIR (Findable, Accessible, Interoperable, Reusable) principles are provided. Finally, commentary on the bottlenecks and pitfalls frequently encountered when attempting to establish a co-culture with sugar-secreting cyanobacteria and a novel heterotrophic partner is included. This protocol provides a resource for researchers attempting to establish a new pair of co-cultured microbes that includes a cyanobacterium and a heterotrophic microbe.

This protocol provides a comprehensive guideline for the setup and quantitative monitoring of co-cultures, including photoautotrophic sugar-secreting cyanobacteria and heterotrophic yeasts.

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Summary

Explore More Videos

Chapters in this video

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Visualizing Yeast Organelles with Fluorescent Protein Markers

Yeast Colony Embedding Method

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

4D Microscopy of Yeast

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure