Yeast Colony Embedding Method

Summary

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.

Abstract

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.

Protocol

1. Colony Isolation and Fixation

1. Incubate 300 colonies on agar medium for the indicated time (an isolated colony should be 1-2 mm in diameter).
2. Remove colony (face up) and underlying medium using a narrow spatula.
3. Place several drops of 2% agar 42°C on a microscope slide using 1 mL pipetman tip and immediately place colony on agar face up before it solidifies.
4. Place several drops of 2% agar 42°C on colony and allow to solidify.
5. Wear gloves for all steps through the remainder of this protocol
6. Trim block with razor blade and place them in a 3.5 mL borosilicate screw-cap vial containing 2% paraformaldehyde/2% gl....

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 amin–thanol	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