The overall goal of this procedure is to embed yeast colonies. This is accomplished by first removing a colony and the underlying agar from an agar plate, placing it on several drops of molten agar and quickly covering it with more molten aer. Next excess AER is trimmed from the colony to make it small enough to fit into a mold.
The AER embedded colony is then fixed, stained, dehydrated, and finally infiltrated with spurs resin. The block is then placed in a mold with additional spurs resin incubated until hardened and then sectioned. Ultimately, results can be obtained that show the distribution of cell types within a yeast colony through light or electron microscopy.
The main advantage of this technique over existing methods like scanning electron microscopy, is that it allows the internal structure of the colony to be determined. This method can help answer key questions in the field of microbial development, such as the distribution of cell types within colonies. This method provided insight into the structure of croce CAC colonies, but it is likely that it can also be applied to other microorganisms.
Visual demonstration of this method is critical, since handling and embedding of colonies may be difficult to learn from a written protocol. The idea for this method was inspired by some genetic data we published in 2002 that suggested that spores were not evenly distributed in colonies. We adapted a method for sectioning colonies that was developed by engelberg in the fin lab.
So let's get started. Begin by incubating Wild S visi yeast on auger plates at 30 degrees until approximately 300 colonies are present. Using a narrow spatula, remove colonies of one to two millimeter diameter, one at a time from the plate.
Then using a one milliliter pipette man tip place several drops of 2%agar at 42 degrees Celsius on a microscope slide, and immediately place the colony on agar face up before the AER solidifies. Place several more drops of agar on the colony and allow the agar to solidify ensure that the entire colony, including the top of the colony, is encased in agar. In all subsequent protocol steps with a razor blade.
Trim the AER around the colony and place the AER block containing the colony. In a 3.5 milliliter bo silicate screw cap vial containing 2%paraform aldehyde, and 2%glutaraldehyde fixative colonies must be processed, one at a time to prevent drying, but up to 10 colonies can be placed in the same vial. Leave the colonies for seven days at four degrees Celsius.
After one week, remove the fixative and wash the agri colonies twice, using approximately 1.5 milliliters of 0.15 molar sodium. Cate at pH 7.2 and incubate on ice after each wash for 15 minutes without agitation. Following this wash twice more.
Using 1.5 milliliters of one X OS buffer, composed of 100 millimolar mono potassium phosphate, 10 millimolar magnesium chloride at pH 6.0, incubating on ice for five minutes after each wash. Next, using the same vial, perform the osmium treatment and washes listed here and in the written portion of this protocol. Then again, using the same vial, perform the stepwise dehydrations listed here and in the accompanying written protocol early the next morning.
Prepare spurs reagent as shown here and in the accompanying written protocol. Immediately wash the agri blocks five times for 10 minutes each with 1.5 milliliters of 100%room temp ethanol. After the last ethanol wash, remove only enough ethanol so that agri block remains covered.
Using pasture pipette, place approximately 0.5 milliliters of spurs reagent into the vial and rotate on a wheel at low speed for 15 minutes. At room temperature after rotation, allow the vial to stand for 30 minutes. Next, remove the solution completely.
Then add 1.5 milliliters of spurs reagent to cover the agri block. Again, rotate the vial on a wheel for 15 minutes at room temperature and allow to stand for 30 minutes. Repeat this wash.
Step three more times. After the final 30 minutes, remove spurs reagent and add fresh spurs reagent to cover the blocks. Allow the agri blocks to stand at room temp for four hours.
Replace the spurs reagent and rotate overnight. The following morning, replace spurs reagent and leave the vials rotating until the following day. On the next morning, place spurs reagent in numbered silicone molds to just cover the bottom of the molds.
Next place the agri blocks into the molds and incubate at 60 degrees Celsius for four hours. After four hours, top off the molds with more spurs, reagent, and incubate at 60 degrees Celsius overnight. Remove the blocks from the mold for sectioning.
An example of a light micrograph of a section through the center of a colony of Wild S Visia yeast colony is shown here. This yeast is a wild type strain isolated from tree exudates. The colony was incubated for six days on YNA medium prior to sectioning.
In this image, asai, pseudo hyphy and ovoid yeast are easily distinguishable and the region of the colony invading the underlying agar is also apparent. Open arrows indicate representative asai filled arrows indicate representative ovoid vegetative cells filled arrowhead indicate chains of elongated cells and si. This image is an example of an electron micrograph from a thin section of a laboratory strain of yeast SH 1 0 2 0, and W 3 0 3.Background.
The colony was incubated for six days on YNA medium prior to sectioning. The image shows a region of the colony containing a high frequency of sporulated cells. An arrow indicates the bilayer structure of the spore wall.
In part A, we show a section of a four day colony with a grid composed of 15 rectangles superimposed on the image in part B.The frequency of spore related cells in each rectangle is graft with the 15 bars corresponding to the 15 rectangles shown in part A.The data are the mean and standard error of four independent four day colonies In part C, the means and standard errors for four independent eight day colonies are shown. Once mastered, this technique can be performed in two weeks with one full day and several one to three hour days. While attempting this procedure, it's important to remember to add the solutions immediately so that the blocks do not dry out Following this procedure.
Other methods, like immuno electron microscopy could possibly be used to determine the distribution of cells that express particular proteins after its development. This technique allowed us to look at not just the patterning of sporulate cells, but also the patterning of pseudo hyphal cells in croce CCI colonies. After watching this video, you should have a good understanding of how to embed yeast colonies for sectioning.
Don't forget that many of the microscopy reagents used in this protocol can be extremely dangerous. Please remember to use a fume hood, wear protective clothing and dispose of the reagents properly. Okay, that's it.
Thanks for watching. Good luck with your experiments.
