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...

<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>

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 alternativ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <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 ...

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

Research

JoVE Journal

Biology

8.8K visualizações.  Fred Hutchinson Cancer Research Center. Nós apresentamos um protocolo para congelamento e cryosectioning comunidades de leveduras para observar padrões internos de células fluorescentes. O método baseia-se na fixação de metanol e OCT-incorporação de preservar a distribuição espacial das células sem inactivar proteínas fluorescentes dentro de uma comunidade.