School of Biological Sciences, University of Missouri - Kansas City
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Piccirillo, S., Honigberg, S. M. Yeast Colony Embedding Method. J. Vis. Exp. (49), e2510, doi:10.3791/2510 (2011).
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 μ) useful for light microscopy and thin sections (0.1 μ) 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.
1. Colony Isolation and Fixation
2. Washes and Osmium Treatment
3. Wash and Dehydration
4. Spurr's Preparation
5. Spurr's Infiltration
7. Representative Results:
The broad relevance of measuring pattern formation in yeast and other microorganisms have been reviewed previously 1. Of particular note is the increased functionality of organized microbial colonies relative to disorganized communities.
The described method is a modification of a method for embedding larger specialized colonies 2, and a short description of our modifications has been published 3.
An example of a light micrograph of a section through the center of a colony of wild S. cerevisiae yeast colony is shown in Figure 1. Asci, pseudohyphae and ovoid yeast are easily distinguishable, and the region of the colony invading the underlying agar is also apparent.
An example of an electron micrograph of a laboratory strain of yeast (W303 background) is shown in Figure 2. The image is from a region of the colony containing a high frequency of sporulated cells.
Figure 1. Light microscopy of a wild yeast colony. This yeast (YPS133) was isolated recently from tree exudates 4. The colony was incubated for 6 days on YNA medium 5 prior to sectioning. Open arrows indicate representative asci, filled arrows indicate representative ovoid vegetative cells, filled arrowheads indicate chains of elongated cells (pseudohyphae) and asci.
Figure 2. Electron microscopy of a laboratory yeast strain. The colony of SH1020 (W303 background) was incubated for 6 days on YNA medium prior to sectioning 3. Image shows a region of the section with a high frequency of asci. An arrow indicates the bi-layer structure of the spore wall.
Figure 3. Quantification of the distribution of sporulated cells in colonies: A) a grid containing 15 adjoining rectangles stacked along their long side is superimposed on the central region of an image of a colony section. The grid is scaled, while keeping its proportions constant, to just covers the top and bottom of the colony. (B & C) The fraction of sporulated cells in each rectangle relative to the total cells in that rectangle is determined by visual inspection of images from 4-day old (B) and 8-day old (C) colonies. For example, the rectangle at the top of the colony (labeled "1") corresponds to the left side of the graph. Mean of 4 colonies is shown; the error bars display the std. error.
The method presented reveals the interior structures of colonies. Because the method is effective in determining patterns of cell types in a range of S. cerevisiae strains with different colony morphologies, and also in a related species S. paradoxus 5, the method is also likely to work on a wide range of fungi and other microorganisms.
One critical step for the success of the method is to ensure that the entire colony, including the top of the colony, is encased in agar throughout the protocol. Whether colonies are completely encased in the agar can be determined after sectioning for light microscopy and staining with toluidine blue. When stained sections are viewed by light microscopy, the agar medium is stained slightly darker than the embedding medium. Because agar shrinks during the dehydrations steps, in order to recover the entire colony be sure to: 1) add 4-5 drops of agar to reliably cover the colony in step 1.2, and 2) trim the agar only on the sides, not on the top and bottom, and only trim enough to allow it to fit within molds in step 1.5.
A second stage of the protocol that is critical for optimal sections involves the hardness of the block of embedding medium. Typically blocks are examined after overnight incubation, by removing them from the molds. If the blocks are still flexible when held between both hands and flexed, they are probably not hard enough for sectioning. In this case they are returned to the incubator at the same temperature for an additional 48 hours and retested as above.
The significance of being able to detect patterns of cell types within microbial colonies is that these patterns likely reflect a functional organization of cells into communities. Indeed, microbial patterning may reflect ancient and fundamental mechanisms by which organisms of the same species interact. Together with methods for monitoring patterns of gene expression in colonies 3,6, the ability to detect patterns of cell differentiation in these communities can help to test potential mechanisms of microbial pattern formation.
No conflicts of interest declared.
Research was funded by NIH 1R15GM094770.
|Osmium tetroxide||Electron Microscopy Sciences||RT 19152|
|Silicone embedding molds||Fisher Scientific||NC 9975029|
|Cycloaliphatic epoxide resin||Electron Microscopy Sciences||RT 15004||ERL 4221|
|Epoxy resin||Electron Microscopy Sciences||RT 13000||DER 736|
|Nonenyl Succinic Anhydride||Electron Microscopy Sciences||RT 19050||NSA|
|2-Dimethyl aminoethanol||Electron Microscopy Sciences||RT 13300||DMAE|
|Mounting media||KPL Inc||71-00-16|
|Rotating wheel||Ted Pella, Inc.||Pelco 1055|
|Microtome||Leica Microsystems||Ultracut S|