Materials Included In Kit

Additional Materials Required

Prelab Preparation

Preparing Lysol Solution

1. Prepare 1 bottle of working-strength Lysol for each lab station.
2. Prepare enough 10% Lysol solution to fill each bottle about half way. For example, add 10 mL of Lysol to 90 mL of DI water to make 100 mL of a 10% Lysol solution.

Agar Media Preparation 
The recipe will make approximately 20 plates if poured to a thickness that just covers the bottom of each dish. Prepare the agar and pour the plates several days in advance of the lab.

1. Add 500 mL of DI water to the 1-L Erlenmeyer flask. Begin heating the water using a stirring hot plate.
2. Add the stir bar and stir vigorously while adding small amounts of the CGY agar to the DI water. Once all of the CGY agar has been added, reduce the speed of the stir bar.
3. Insert the foam plug into the mouth of the Erlenmeyer flask.
4. Stir on a moderate setting while slowly heating the agar until it just begins to boil. Note: The solution will start out cloudy and then become a clear amber color as it heats up.
5. Autoclave the container for 15 minutes at 121 °C with 15 lbs. pressure. Most autoclaves set for 15 minutes will do the rest automatically. Caution: Read and follow instructions for specific pressure cooker or autoclave. After the sterilization is finished, pour CGY agar into plates or store in the refrigerator.

Pouring Plates 

1. The CGY agar should be about 55 °C to pour properly.
2. Disinfect the work area with a disinfecting solution such as a freshly prepared 10% bleach or Lysol solution. Be sure the work area will hold all 20 plates so they can be poured without stopping.
3. Wash hands with antiseptic soap.
4. Remove sterile Petri dishes from plastic holding sleeve. Note: Do not open the dishes.
5. Place the dishes right side up on the work surface as indicated in Figure 6. {11048_Preparation_Figure_6}
6. Remove the foam plug from the Erlenmeyer flask and flame the top of the container by passing it through a blue Bunsen burner flame. Rotate the bottle to be sure all sides of the opening are flamed.
7. Tip the lid of a Petri dish and pour in liquid until the bottom is just barely covered—approximately 15 mL (Figure 7). {11048_Preparation_Figure_7}
8. Repeat step 7 for the remaining Petri dishes. Work quickly but avoid creating drafts.
9. Once all plates are poured, allow the CGY agar to solidify. This should take 5 to 10 minutes depending on the ambient temperature.
10. After the CGY agar solidifies, turn the plates over so that agar is on the top as shown in Figure 8. Store the unused plates in the refrigerator. {11048_Preparation_Figure_8}

Subculturing the Sordaria 
Please read the complete procedure before proceeding as there are some differences between culturing Sordaria and culturing bacteria, which may affect results.

1. Disinfect the work area with a 10% Lysol solution. Be sure the work area will hold all 20 plates so they can be poured without stopping.
2. Wash hands with antiseptic soap.
3. Place two of the CGY agar plates right side up on the work surface.
4. Flame the tip of an inoculating loop within the blue part of the Bunsen burner flame until it glows red. Hold the loop still while it cools.
5. Using the hand holding the loop, remove the top from the wild type Sordaria culture and flame the top of the culture tube by passing it though the flame. Rotate the tube to be sure the entire top of the tube is sterilized.
6. Insert the sterile loop into the stock sterile culture. Remove a small portion of the Sordaria and agar. Note: The traditional drag technique used to culture bacteria will not yield enough Sordaria.
7. Remove the loop with the agar section and place it face down onto the center of the CGY agar plate. Label the plate.
8. Repeat steps 4–7 for the second wild type Sordaria subculture plate.
9. Repeat steps 1–8 for the tan mutant Sordaria culture.
10. Seal the four subculture plates with Parafilm. Incubate at 30 °C in a humidified incubator for several days until a lawn of Sordaria has developed.
11. Disinfect the lab table and thoroughly wash your hands.

Safety Precautions

Wear chemical splash goggles and chemical-resistant gloves. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All microorganisms purchased from Flinn Scientific are non-pathogenic. However, to be safe all cultures should be considered pathogenic and destroyed according to Flinn Biological Waste Disposal Type I before disposal in the trash. The best way to dispose of fungi on agar plates is to autoclave them in a heat-stable biohazard bag. If an autoclave is not available, saturate the agar plates with a 10% Lysol solution. Allow plates to sit for 24 hours before disposing in the trash.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs, or for 15 groups of students. Both parts of this laboratory activity will require between 5 and 10 days to complete. One full lab period is necessary to prepare the cross plates. A second full lab period is necessary 5–10 days later for the collection of perithecia and the counting of asci. The prelaboratory assignment may be completed before coming to lab, and the data compilation and calculations may be completed the day after the lab.
• Students should be proficient with microscopes, wet-mount slide preparation and aseptic technique prior to beginning this laboratory.
• Locating mature perithecia before they rupture and release ascospores is essential to the success of this laboratory. It may be necessary to check the plates before or after school to ensure success. When the perithecia are mature transfer the cross plates into a refrigerator to slow maturity.
• The technique of good squash slide preparation can be challenging for some students. Allow successful students to share tips and techniques with other students. Keep good preps for a few weeks by painting around the edge of the cover slip with clear nail polish.
• Counting Crossing Over (Catalog No. FB1973) can be used as a substitute simulation activity in case a student is absent or is sensitive to mold.

Further Extensions

Concepts and Curriculum Framework for AP^® Biology

Big Idea 2: Biological systems utilize free energy and molecular building blocks to grow, to reproduce and to maintain dynamic homeostasis.

Enduring Understandings
2A3: Organisms must exchange matter with the environment to grow, reproduce, and maintain organization.

Big Idea 3: Living systems store, retrieve, transmit and respond to information essential to life processes.

Enduring Understandings
3A1: DNA, and in some cases RNA, is the primary source of heritable information.
3A2: In eukaryotes, heritable information is passed to the next generation via processes that include the cell cycle and mitosis or meiosis plus fertilization.
3A3: The chromosomal basis of inheritance provides an understanding of the pattern of passage (transmission) of genes from parent to offspring.
3C2: Biological systems have multiple processes that increase genetic variation.

Learning Objectives 

• The student can make predictions about natural phenomena occurring during the cell cycle (3A2 & SP 6.4).
• The student is able to construct an explanation, using visual representations or narratives, as to how DNA in chromosomes is transmitted to the next generation via mitosis, or meiosis followed by fertilization (3A2 & SP 6.2).
• The student is able to represent the connection between meiosis and increased genetic diversity necessary for evolution (3A2 & SP 7.1).
• The student is able to evaluate evidence provided by data sets to support the claim that heritable information is passed from one generation to another generation through mitosis, or meiosis followed by fertilization (3A2 & SP 5.3).
• The student is able to construct a representation that connects the process of meiosis to the passage of traits from parent to offspring (3A3 & SP 1.1, SP 7.2).
• The student is able to construct an explanation of the multiple processes that increase variation within a population (3C2 & SP 6.2).

Science Practices
1.1 The student can create representations and models of natural or man-made phenomena and systems in the domain.
1.2 The student can describe representations and models of natural or man-made phenomena and systems in the domain.
5.3 The student can evaluate the evidence provided by data sets in relation to a particular scientific question.
6.2 The student can construct explanations of phenomena based on evidence produced through scientific practices.
6.4 The student can make claims and predictions about natural phenomena based on scientific theories and models.
7.1 The student can connect phenomena and models across spatial and temporal scales.
7.2 The student can connect concepts in and across domains to generalize or extrapolate in and/or across enduring understandings and/or big ideas.

Answers to Prelab Questions

1. In order to calculate the number of map units between the centromere and the gene at least 100 asci are counted. Why is it necessary to count so many asci?

   The number of asci counted must be statistically significant, otherwise the data will be skewed.

2. Calculating map units was the only way to determine the physical location of a gene along a chromosome. Now chromosome mapping uses special enzymes to clip the DNA of the chromosomes into chunks which are sequenced using advanced instrumentation and computer programs. Map unit calculations still provide important information to geneticists. Speculate why map unit calculations are still important.

   Map unit calculation help scientists place the gene map on the correct chromosome in the correct general location.

Sample Data

Answers to Questions

1. Take the sum of the tally marks for each genotype. Record each result in the Total column.

   See data table for an idealized answer. Student answers will vary.

2. Determine the total number of non-crossover asci counted.

   Based on the sample data in the table the number is 74. Student answers will vary.

3. Determine the total number of crossover asci counted.

   Based on the sample data in the table the number is 26. Student answers will vary.

4. Determine the total number of hybrid asci counted.

   Based on the sample data in the table the number is 100. Student answers will vary.

5. Determine the map distance between the gene for spore color and the centromere using Equation 1. Report the result in map units. However, keep in mind that each ascus contains 8 spores because the four haploid spores underwent an additional mitotic event after meiosis. To account for this, the map distance found in Equation 1 needs to be halved (Equation 2).
   {11048_Answers_Equation_1}
   13/50 x 100 = 26 map units
   {11048_Answers_Equation_2}
   26/2 = 13 map units
6. Was the number of each type of crossover phenotype observed relatively constant or equal? Explain why you would expect these numbers to be constant?

   Yes, each crossover phenotype occurred in similar numbers. This is due to the random segregation of each allele during meiosis.

7. A similar technique can be used to determine the distance between two genes or a different gene and the centromere. In this laboratory a color mutation was used as the gene of interest. What is the benefit for using a color mutant gene of learning about map units?

   Observations are easy if the characteristic of interest is a visible trait.

References

Olive, L. S. Genetics of Sordaria fimicola; American Journal of Botany, 1956, 43, 97–107.

Cassell, P., Mertens, T. R., A Laboratory Exercise on the Genetics of Ascospore Color in Sordaria fimicola; The American Biology Teacher, 1968, 30, 367–372.

The Tree of Life web project http://tolweb.org/Ascomycota (accessed August 2010).

Biology: Lab Manual; College Entrance Examination Board: 2001.

Introduction

Ascomycota are a diverse group of fungi containing the familiar single-celled baker’s yeast, the complex morel mushroom, and the deadly Aspergillus flavus. In fact, 75% of all fungi are grouped as Ascomycota. Geneticists have altered one particular species of Ascomycota, Sordaria fimicola, for use in studying crossing over during meiosis.

Concepts

• Meiosis
• Genetics
• Mitosis
• Crossing over
• Centromere

Background

Crossing over occurs during prometaphase I of meiosis. During crossing over, homologous pairs of chromosomes exchange sections of DNA that contain the same genes. Therefore, the exact genotype of the new offspring will vary from that of its parents (see Figure 1). It is important to note that crossing over does not have to occur during each generation, nor does it always take place at the same point of exchange. Over time, however, crossing over leads to a greater variety of genes in a population and contributes to a diversity of characteristics and an overall stronger population. This strength is then reflected in the ability of the population to adapt to changes in the environment and also to evolve.

Meiosis involves two cellular divisions, meiosis I and meiosis II. In meiosis I the chromosomes condense, replicate, crossover and divide in two. In meiosis II, the chromosomes do not replicate again. Instead each chromosome is split in half through the centromere leaving one copy of each gene in each haploid cell. In the fungi kingdom, meiosis occurs in specialized fruiting bodies. In the group Ascomycota this specialized fruiting body is called an ascocarp or perithecium. The frequency of crossing over is interesting in studying genetics because it allows scientists to map genes and estimate the distance between two genes or between a gene and the centromere of the chromosome. The daughter cells are called ascospores or, in more general terms, spores. The daughter cells are all contained within a single tube-like structure called an ascus (plural = asci). The structure and properties of the ascus make Sordaria fimicola useful for studying crossing over.

Many Ascomycota, like S. fimicola, spend most of their time as haploid cells. Numerous clone copies of each haploid cell unite to form thread-like hyphae. Small holes between cell walls allow the sharing of nutrients and water between the cells of each hypha. Masses of hyphae intertwine to form mats of fungi. One of the reasons that fungi spread so easily is that these haploid hyphae are able to break off and generate a new organism anywhere nutrients are available. S. fimicola grows on rotting vegetation or dung in the wild making it a common mold in the environment. In the lab, S. fimicola is easily cultured on agar plates. If no nutrients are available, the fungus is able to form haploid spores using asexual reproduction or sexual reproduction. The spores can be dispersed in the wind or settle into the soil until conditions improve.

During sexual reproduction hyphae of different haploid S. fimicola come into contact allowing cells in the hyphae to fuse and form a single cell with two nuclei, one from each individual. This fused cell is called a dikaryon. The dikaryon is not considered diploid since the two nuclei are from separate fungi and the nuclei are not fused together. The dikaryon cells undergo multiple rounds of mitosis to form a mass of cells. This mass of cells can exist for years without undergoing fusion of the nuclei. Sexual reproduction occurs when some of the dikaryon nuclei fuse. After fusion the fruiting body forms and meiosis occurs, creating the asci and ascospores of the next set of haploid cells.

The ascospores form inside the tight confines of the tube-like asci. The ascospores actually line up in order based upon which cell produced that particular ascospore. In 1956, a geneticist named Lindsay S. Olive (1917–1988) published an article about crossing over in S. fimicola. Dr. Olive used ultraviolet light to cause mutations in the genes of S. fimicola. After numerous trials Dr. Olive produced a mutation in the gene that produces the pigment in the ascospore. The production of the black pigment is either greatly reduced or completely repressed in the mutated strain of sordia. A reduction in the amount of black pigment results in gray spores. An absence of black pigment results in tan ascospores. By collecting the gray or tan ascospores Dr. Olive was able to produce true breeding fungi much like Mendel’s peas.

Collecting the ascospores is easy because the fruiting body produced by S. fimicola is shaped like a vase (see Figure 2). The vase-shaped perithecium is produced on a dikaryon stalk above the dikaryon mass of cells. Within the perithecium each ascus lines up with the top opening of the perithecium and the ascospores are ejected out into the wind for dispersal. In order to isolate and identify ascaspores, it is important to collect the perithecium just before the asci eject the spores. Wet-mount microscope slide preparations of the perithecium result in the asci spreading out like spokes on a wheel, and the ascospores become visible for analysis. The distance between the centromere and the gene that codes for the black pigment can be determined by counting the ascospores within a population of asci. This distance is called the map distance and is reported or measured in terms of map units. A map unit is an arbitrary unit of measurement where one map unit corresponds to 1% crossover. The likelihood of crossover occurring between two genes on the same chromosome increases as the distance between the genes increases. Similarly, a gene is more likely to crossover if the gene is not adjacent to the centromere of the chromosome. By definition, the number of map units between two genes or between the gene and the centromere is equivalent to the percent of genes that undergo crossover. In order to count the number of crossing over events a culture of wild type (black) S. fimicola and a culture of tan mutant S. fimicola are grown adjacent to each other in a culture dish. Recall that each ascospore can be tracked back to the parent chromosome. The pattern of black and tan ascospores shows whether crossing over occurred during meiosis (see Figure 2). Note that the diagram of the asci indicates eight ascospores in each ascus, not the expected four cells. With S. fimicola each of the four haploid daughter cells undergoes a single mitosis after the end of meiosis II. So each daughter cell produces a clone of itself. These clones reside next to each other within the ascus. If the cells come from parents with identical pigment genes the ascus will contain eight spores that are the same color whether black or tan. If the cells come from parents with each pigment type but crossing over did not occur the spores will appear as four black wild-type and four tan mutant spores (4b:4t). If crossing over between a black wild-type and a tan mutant occurred during meiosis I the four spores will have one of two possible patterns. Patterns of 2:2:2:2 and 2:4:2 are possible. Each of the numbers can be either tan or black. This is written out as 2b:2t:2b:2t or 2t:2b:2t:2b and 2b:4t:2b or 2t:4b:2t.

Experiment Overview

The distance between the pigment gene and the centromere will be determined by preparing crossover culture plates of black and tan Sordaria. The number of each asci phenotype will be noted followed by the calculation of the map distance.

Materials

Prelab Questions

1. In order to calculate the number of map units between the centromere and the gene, at least 100 asci are counted. Why is it necessary to count so many asci?
2. Calculating map units was the only way to determine the physical location of a gene along a chromosome. Now chromosome mapping uses special enzymes to clip the DNA of the chromosomes into chunks which are sequenced using advanced instrumentation and computer programs. Map unit calculations still provide important information to geneticists. Speculate why map unit calculations are still important.

Safety Precautions

Sensitivity may occur among individuals with severe mold allergies. Use caution when working with flames and heated inoculating loops. Follow aseptic technique throughout this lab and disinfect the work surfaces before and after conducting each part of this lab. Wear chemical splash goggles and chemical-resistant gloves. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Part I. Making Cross Plates

1. Read every step of this procedure before proceeding. It is essential that aseptic technique be used at every step of the activity.
2. Label the top of the CGY plate with the group name. Write as small as possible along the edge of the lid so the label will not interfere with viewing the culture in steps 17–18.
3. Turn the CGY plate over and mark an X approximately ⅓ of the way from one edge along the center diameter of the plate. Place a second X approximately ⅓ of the way from the opposite edge of the plate (see Figure 4). Ensure that the Xs are visible when viewing the plate lid-side-up. These Xs will be the location where the Sordaria plugs will be placed.
   {11048_Procedure_Figure_4}
4. Use the Lysol solution to disinfect the lab table.
5. Wash your hands thoroughly with soap and water before you begin the activity.
6. Light the flame of the Bunsen burner. Adjust the burner so a blue flame exists in the center of the flame.
7. Heat the inoculating loop in the burner flame until it glows red (see Figure 5). Allow the inoculating loop to cool. Note: Do not wave the inoculating loop in the air to cool it.
   {11048_Procedure_Figure_5_Flaming an inoculating loop}
8. Lift the lid of a Sordaria culture as little as possible.
9. Use the inoculating loop to cut a 0.5-mm cube of agar from an area of the Sordaria culture that has obvious growth on it.
10. Use the inoculating loop to lift the cube of agar from the Sordaria culture.
11. Lift the lid of the marked CGY agar plate as little as possible.
12. Place the cube of Sordaria, fungus side DOWN, on one of the two Xs marked in step 3.
13. Repeat steps 5–12 with the second Sordaria culture placing the plug on the second X.
14. Seal the CGY plate with Parafilm.
15. Disinfect the lab table and thoroughly wash your hands with soap and water.
16. Invert the plates and incubate at 30 °C for 5–10 days.

Part II. Observing Asci

17. Check the CGY plate daily to ensure the plate is not contaminated. After 5 days begin checking the perithecium to determine maturity. If no mature perithecium are present return the cross plate to the incubator for another day.
18. Use the stereoscope to look at the perithecium along the center line of the cross plate. It is not necessary to lift the lid. Mature perithecium are black with a shape that resembles a vase. Note: The center perithecium will show crossing over while those along the outside of the plate will remain either all wild type or all tan mutants depending upon the side of the plate.
19. Wet a sheet of folded paper towel with Lysol solution. Clean the dissecting needle with the wet paper towel. Allow it to air dry.
20. On a separate sheet of paper towel place several clean microscope slides. Place the 50% glycerin solution, the cover slips, and the pencil near the slides.
21. Place a single drop of the glycerin solution onto one of the microscope slides.
22. Through the closed lid, determine which perithecia are mature.
23. As quickly as possible remove the lid of the cross plate and use the dissecting needle to remove several mature perithecia from the culture. Replace the lid immediately.
24. Transfer the perithecia into the drop of glycerin solution.
25. Place the cover slip onto the drop of glycerin solution.
26. Use the eraser side of the pencil to gently press down on the cover slip. Note: It may take some practice to determine the correct amount of pressure to exert. Too little pressure and the perithecia will not rupture. Too much pressure and the ascospores will rupture out of the asci or the cover slip will break. If this step is difficult, practice using perithecia from areas where crossing over is less likely to occur until the technique has been mastered.
27. Locate the ruptured perithecia using low power on a compound light microscope. When prepared properly, the asci will form an asterisk pattern next to the ruptured perithecia.
28. Use higher power to determine the pattern of ascospores in each ascus. Begin at the 12 o’clock position and work in a clockwise rotation to ensure each ascus is counted. Use tally marks on the Mold Crossing Worksheet to keep track of the number of each pattern seen.
29. Repeat steps 21–28 until a total of 100 asci have been counted.

Student Worksheet PDF

11048_Student1.pdf