Teacher Tips:

The Yeast Life Cycle experiment in this manual uses haploids that are cream-colored (HBT) and pink (HA2) to make the sexual cross. Those two strains illustrate that the cream-colored phenotype is dominant over the pink phenotype. It is possible to analyze the ascospores from that cross but you will have to determine the trp5 genotype present in HBT by plating the cells on MV+ade medium. (TRP5 will grow, trp5 will not grow)

Rather than having the whole class make the cross (HA1 X HB2) suggested in this experiment you may wish to have several students perform the Getting Ready steps as a class demonstration. This will offer the opportunity for the whole class to review the steps of the yeast sexual life cycle. One plate of asci will supply enough ascospores for the entire class to do this spore analysis experiment. Getting Ready: 1st Day - 8th Day: 30 min
It takes less than 10 minutes to perform each of the yeast transfers in these Getting Ready steps. You will need to prepare the agar plates in advance.

1. To avoid contamination it's usually best to limit student access to your master collection of yeast strains. You may wish to subculture the strains on one plate of YED and then use that plate as the source of strains for student work.

3. The diploid cells will be cream-colored because each cell has at least one functional copy of each adenine gene. (See Genetics of Baker's Yeast in this manual) HA1 (ade1 ADE2); HB2 (ADE1 ade2); HA1 X HB2 (ade1/ADE1 ADE2/ade2)

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This is sufficient to test whether the overall segregation frequency is as expected. (75% pink, 25% cream-colored) Chances are, however, there will be a large excess of cream-colored, adenine-independent colonies. This is because the sample will include some unsporulated diploids. If the sporulation step doesn't work well the cream-colored diploid colonies could account for most of the isolated cream-colored colonies that you count. Longer digestion in snail enzyme will kill some of the diploids, but if you go too long it will kill spores, too. To get a better estimation of the segregation frequency you must determine what fraction of your isolated cream-colored colonies are haploid. Digestion of Asci, Plating Cells and Counting Colonies: 9th Day:
1. The snail enzyme used in this experiment is Glusulase, a -glucuronidase/sulfatase prepara$ of snails, Helix pomatia. A crude solution is adequate. Sigma Chemical Co. is one possible supplier. It is usually best to dilute the crude solution with sterile water. A one 2.to twenty dilution usually works well. (1 part glusulase in 19 parts sterile water) Because the activity of these crude enzyme solutions may vary you may wish to experiment with several different dilutions. Store the unused solutions in the refrigerator.

2. Toothpicks are sterile from the box. If you use an inoculating loop you will need to supply a flame to sterilize the loop each time it is used. (See Laboratory Methods, Using Toothpicks and Inoculating Loops)

3. Use a sterile loop to transfer a small amount of the suspension to a microscope slide. You may need to add a small amount of water to the yeast suspension before you add the coverslip. (See Laboratory Methods Using Toothpicks and Inoculating Loops)

Five to six single colony isolation plates should be enough for each class if the ascus digestion works well. You may wish to have each student make an isolation plate to practice the technique and to increase the number of haploid colonies available for analysis. (See Laboratory Methods, Isolating Single Colonies)

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Comment: You can now go back to your original colony count data from Day 10. Multiply the original cream-colored colony count by the haploid fraction that you just determined to get a more accurate number of cream-colored haploid colonies.

"corrected" cream-colored haploid count = original cream-colored count * haploid fraction

When you use the "corrected" cream-colored colony count and the original pink colony count does your phenotype segregation frequency equal the expected 75% pink and 25% cream-colored?

When you pool the class data does the phenotype segregation frequency come closer to the expected frequency of 75% to 25% ?

If there is a reasonable number of red colonies, indicating that the sporulation worked (remember that theoretically 75 percent could be red), then it is instructive to determine the mating type and adenine genotype of some of the spores. Analysis of Cream-colored Colonies:

11th Day:
1. Make a 2 to 3 centimeter streak from each colony. You may wish to number each source colony on the bottom of the source plate and then record those numbers on the YEKAC plate. You will then be able to go back to the source plate when you identify which cream-colored colonies are haploid. (See The Yeast Life Cycle section of more information on sporulating cells)

15th Day:
1. (See Laboratory Methods Microscopic Examination of Yeast and the Yeast Life Cycle section)

2. Haploid fraction
If the original sporulation frequency was low it is possible for an individual student to get 0 haploids in their sample of 5 to 10 cream-colored colonies.

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Determining the genotypes of ascospore colonies:

The possible genotypes of colonies from the digested sporulation mixture are the parental diploid genotype and the eight possible haploid genotypes in Table I. Any red colony must be haploid, but the cream-colored ones could be either haploid or diploid. The red spore strains can be tested for ade1 or ade2 at the same time that they are tested for mating type. This can be done by using the mating and complementation test using four tester strains: a ade1, ` ade1, a ade2, and ` ade2. Each red spore strain is crossed with each of the four testers and then tested for mating by complementation on MV. Neither the red spore strain nor the tester strains can grow on MV. If they mate and complement the resulting diploid will be able to grow on MV. Each should mate and complement with no more than one tester; a double ade1 ade2 mutant will not complement with either. Its mating type, and the mating types of the cream-colored, adenine-independent spores, can only $ in the mating mixture.

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Mating of Strains
: 1. Be sure to use a fresh sterile toothpick each time you start working with a new strain.

2. Remember to use a fresh sterile toothpick for each of the mating mixtures when you stir each mixture together.

3. The mixtures from red spore colonies will be transferred to MV.

4. You may wish to use a video microscope to show the class asci so they will know for sure what to look for in their slides.

5. This test determines the mating type. The cream color of the haploid strains indicates the ADE1 ADE2 adenine independent genotype. If you wish to confirm the genotype you may wish to plate the haploid strains on MV medium.


This completes a total analysis of the segregation of three pairs of heterozygous markers. The results can be compared statistically with the expected results by using the Chi-square test. For this to be valid it is important that the sample of spores not be biased for or against red colonies. The best way is to test all the colonies that can be picked from each plate. It may be useful to organize a group effort and pool the results.

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Last updated Friday July 11 1997