Thimons1

Introduction:

Sordaria fimicola belongs to the fungal class of ascomycetes, or sac fungi. (Carolina

Biological Supply Company) These microscopic fungi are found in the fecal matter of

herbivores. Sordaria has a life span around 7 to 12 days and is easily formed. Sordaira produces

perithecia reproductive structures in many species of mushrooms. The color of Sordaria varies,

but is usually found in a dark brown shade, other mutant colors would include a tan or greyish

color swirled and infused into it. (Pearson: The Biology Place) Sordaria belongs to the Sordaria

family, sordariacease. During the life cycle of sordaira fimicola, it is most commonly and most

often seen in the haploid cell form. In the first division of Meiosis, crossing over occurs and in

regards to Sordaria, it reproduces sexually due to the genes of two separate asci that undergo the

same process. Sordaria can be for the ecosystem because the dung the fungi is found in get

recycled back into the soil to provide nutrients for it. The life cycle of Sordaria starts off with a

single haploid cell. This haploid cell then falls to the ground and begins to germinate. The

process of germination is the [organism] coming to existence. (dictionary.com) After Meiosis I,

Mitosis occurs. In Mitosis, haploid cells are transformed into diploid cells. The cells then begin

to expand and stray off which are the mycelia start to form. These two mating types come

together, both the positive and negative sacs. The positive mating type forms ascogonium, a sac

developed in the process. The sac that the negative mating type creates in this process is called

antheridium. The nuclei are contained inside both ascogonium and antheridium. The positive and

negative sacs then form together, which is the process called plasmogamy. He newly fused sacs

create one single sac combining all of the positive and negative types together. This dikaryotic

cell is a cell that has two haploid nuclei left over, that have not been fused together yet. This

eventually branches out, which is creating more cells than previously. The ascocarp, which is the
 Thimons2

fruiting body of the fungus, is created from these branches. These branches are the dikaryotic

cells weaved together to create a figure that contains many asci in a bunch that appears like a

circle . A new, individual sac is formed and this is called the Ascus, which contains two haploid

nuclei. The process of karyogamy takes place, where the two haploid nuclei fuse to create one

diploid nucleus. Due to Meiosis, four haploid nuclei were created from diploid cells. Mitosis

occurred in this situation so that the four haploid nuclei are able to create eight haploid nuclei

inside of one Sordaria ascus. The nuclei start to burst of the ascus breaking them up. This is

when the life cycle of Sordaria fimicola starts from the beginning and loops back around. There

are two genes that determine ascospore color and its alleles. Each of the two genes has its own

two alleles which are seen in two different forms. They work together to determine color of

fungus. The possible colors of you will find are tan (mutant), gray (mutant), black (wild), and

clear. Both the tan (tn) and the gray (g) are fused with the wild type, or black, (+) to create new

Sordaria femicola. The dependent variable in this lab is the was the rate of crossing over. The

independent variable was the type of fungi.

Materials: (Carolina Biological Supply Company, 1999)

Sordaria fimicola, wild type

Sordaria fimicola, mutant gray
Sordaria fimicola, mutant tan
Bottle cornmeal-glucose-yeast agar
Autoclavable disposable bag
3 bottles Sordaria crossing agar
20 sterile petri dishes
Microscopes
Glass slides and cover slips
Water dropping bottles
Inoculating loops
 Thimons3

Bunsen burner
Boiling water bath
Scalpel or spearpoint needle
Disinfectant such as phenol or 70% ethanol

Procedures: (Carolina Biological Supply Company, 1999)

Preparation of Agar Dishes:

1. Slightly loosen the bottle caps and set the bottles in a boiling water bath to melt the agar.

(Caution: Since the labels may come off the bottles during boiling, it is advisable to mark

the bottle caps with the type of agar contained within.) Make sure the water level is even

with the agar level. Swirl the bottles gently to be sure that all the agar has melted.
2. Cool the agar to 45 degrees Celsius (the bottle should feel comfortable hot to the touch)

by cooling the water bath to that temperature or by letting them sit for several minutes at

room temperature.
3. Wipe down the work surface with a disinfectant such as phenol or 70% ethanol. Wash

your hands.
4. Swirl the bottle of cornmeal-glucose-yeast agar, remove the cap, flame the mouth over a

Bunsen burner for a few seconds, and distribute the contents among six petri dishes. Lift

the lid of the dish just enough to pour in the molten agar. Replace the lid of the lid just

enough to pour in the molten agar. Replace the lid immediately to prevent contamination.
5. Label each dish with the type of agar.
6. Repeat steps 4 and 5 with the Sordaria crossing agar, distributing the remaining agar

among the 14 dishes.

7. After all the agars have been solidified, the dishes may be stored for up to a week at room

temperature or in the refrigerator.

 Thimons4

8. Dispose of the bottles in the autoclavical disposable bag.

Preparation of Stock Cultures:

1. Disinfect the work surface and wash your hands.

2. When ready for use, label two of the cornmeal-glucose-yeast agar dishes wild, two

gray, and two tan.

3. Using aseptic technique, inoculate the dishes with the appropriate culture. Remove the

top from the tube of wild-type Sordaria fimicola, and flame the mouth over a Bunsen

burner for a few seconds. With a flamed, cooled scalpel or spearpoint needle, remove a

portion of the culture containing perithecia (black peppergrain appearance) and transfer to

the middle of a cornmeal-glucose-yeast agar dish. Repeat this procedure to prepare

another wild-type culture.

4. Using the other tubes, follow step 3 to prepare two gray and two tan stock culture dishes.
5. Incubate the dishes for 5 to 7 days out of direct sunlight at room temperature (22 degrees-

25 degrees Celsius) until perithecia have formed at the periphery of the dishes.

During Laboratory 1: Preparing the Crosses:

1. Disinfect the work surfaces. Have the students wash their hands.
2. Label one half of the Sordaria crossing agar dishes +/g and the other half +/tn to

indicate crosses between the wild-type and mutant-gray for wild-type and mutant tan)

strains.
3. Invert the dishes over Figure 1. Using a wax pencil or permanent marker, indicate th

positions of wild type (+) and gray (g) or tan (tn) cultures.
4. Using a flamed, cooled, scalpel or sprearpoint needle, cut the agar in the stock culture

dishes into 0.5 cm cubes. Place the cubes upside down over the indicated positions on the

surface of the crossing agar. Each plate will contain two blocks of the wild-type culture

and two blocks of either tan or gray culture.

 Thimons5

5. Incubate the dishes out of direct sunlight and at room temperature.

6. From 8 days after inoculation until forcible discharge of the spores, genetic data can be

obtained. Usually, the cultures should be ready for microscopic examination in 8 to 10

days, but at cooler temperatures, 14 to 15 days may be required. In order to obtain

accurate data, it is essential that mature ascospores be counted. If it is difficult to

distinguish microscopically between the wild-type and gray or tan spores, the ascospores

are too immature to collect data. Incubate the cross dishes for another day or two and

observe again.

During Laboratory 2: Microscopic Examination

1. Disinfect all work surfaces. Have the students wash their hands. Point out the location of

the autoclavable disposable bag.

2. Provide the students with water dropping bottles, glass slides, cover slips, inoculating

loops, and microscopes.

3. Remove a few perithecia from the cross dishes with a flamed, cooled loop and prepare a

wet mount. Have the students note from which cross plate (+/tn or +/g) they are

removing perithecia. Notice the locations are different for gray and tan hybrid asci.
 Thimons6

Instruct the students to mentally note the position on the dish from which they prepared

their slide. When students locate an area on the dish where hybrid asci are found, they

can share this information with the other class members.

4. Press the cover slip gently using the thumb or an eraser to crush the perithcia and release

the rosettes of the asci. If too much pressure is applied, the ascospore will be forced out

of the asci, making it impossible to collect data. A little practice will perfect the

technique.
5. Using low power, examine the slide and locate rosettes of hybrid asci containing

ascospores of two different colors. The wild-type ascospores appear black, while they

gray and tan spores are a lighter color. Note: Many perithecia contain rosettes with

ascospores of only one color. Preserve in searching until you locate perithecia with hybrid

asci containing spores of two different colors.

 Thimons7

6. After locating a rosette of hybrid asci, use high power to observe the ascospores and

determine if crossing-over has occurred. If crossing-over has not occurred, segregation of

the genes for spore color has taken place during Meiosis I (MI) and the ascospores will be

arranged in a 4:4 ratio. If crossing over has occurred, segregation of the genes for spore

color do not segregate until l Meiosis II (MII) and the arrangement of ascospores will be

either 2:4:2 or 2:2:2:2.

7. Each group should count 100 to 200 asci. Collect class data in Table 1.
8. Chromosome maps for the two mutant genes are constructed by dividing the %MII by 2.
 Thimons8

Results:

Table 1: In Table 1, this shows the number of Sordaria asci that went under the process of

crossing over and how many failed to cross over. For both the tan vs. black cross and the gray vs.

black cross, their distance away from the center of the chromosome which is calculated in map

units.

Strains No. of MI No. of MII Total Asci % MII No. Map Units

Crossed Asci (4:4) Asci (2:4:2 or MII/Total) %MII/2

2:2:2:2)
(g) x (+) 82 141 223 63% 31.5 map

units
 Thimons9

(tn) x (+) 91 147 238 62% 31 map units

In result of the Sordaira lab, the gray gene had the highest percentage, or rate of crossing

over, of Sordaira asci that underwent process of crossing over. A total of 223 asci were seen from

the collection from the petri dishes on a microscope. Out of the 223 asci, 82 did not cross over,

and 141 did cross over. The farther away the nucleus is away from the center of the chromosome,

the more likely it is for it to cross over. This leaves us with 63% for our rate of crossing over,

which why the g gene had a higher percentage than the t gene. The g gene is located 31.5

map units away from the nucleus. As for the tan vs. black gene, a total of 238 asci were seen

under the microscope. Out of the 238 asci, 91 did not cross over and 147 did cross over. The rate

of crossing over for the tan vs. black cross is 62%. The t gene is located 31 map units away

from the center of the chromosome.

Discussion:

The rate of crossing over is very important in figuring out the distance away from the

center of the chromosome. The farther the distance from the center of the chromosome, the more

likely it is to undergo the process of crossing over. Because the gray vs. black cross was located

31.5 map units away from the center of the chromosome, the more likely it was to cross over.

This was reflected in the results of the fungal extraction lab. The g gene had a rate of crossing

over of 63% and the t gene with 62%. One error in the lab that have occurred was not letting

the fungi harvest long enough on the petri dishes. Another error that could have occurred was not
 Thimons
10

crushing the specimen enough or crushing it too much on the slides before placing it under the

microscope. In the procedures, we were told where to scrape the specimen off of the agar

(Carolina Biological Supply Company). We could have accidentally got agar on the slide with

the specimen or extracted it from the wrong spot. When examining the ascospores under the

microscope, some were seen in clumps and easily could have been misread. Over all I think the

results we received were pretty accurate considering the previous errors could have occurred and

effected out results immensely.

Works Cited

"LabBench Activity." Pearson; The Biology Place. N.p., n.d. Web. 29 Apr. 2017.

"Sordaria Lab." Google Slides. Google, n.d. Web. 23 Apr. 2017.

"Welcome to Carolina Biological Supply." Carolina Biological Supply: World-Class Support for

Science & Math. N.p., n.d. Web. 19 Apr. 2017.

Volk, Tom. "Sordaria Fimicola, a Fungus Used in Genetics." Sordaria Fimicola Genetic .

Information. N.p., n.d. Web. 22 Apr. 2017.