Exercise 8 - Meiosis and Crossing-Over

Chromosome positions during meiosis are observed in Ascaris. Students study the life cycle of Sordaria and count the frequency of crossing-over. The class data are summarized in a histogram.

EQUIPMENT	AMOUNT (Class of 24 with 8 groups)
Compound microscopes	1/student
Dissecting microscopes	1/lab
Incubator, 24 ° C	1/lab
Transfer hood (see NOTES)	Instructor use only

MATERIALS

SOLUTIONS

If premade agar plates are not ordered, you will need these materials:

Crossing agar--cornmeal agar (Difco)*, glucose, yeast extract (Brewer's)

PREPARATION

Four Weeks before Lab

Sordaria cultures should be ordered on plates to arrive 14 days before the lab. CBS provides an excellent manual outlining protocols for growth and crossing. Preserved Ascaris can also arrive at this time.

Eleven Days before Lab

If premade plates are not ordered from CBS, you will need to do the following:

1. Crossing agar preparation:

   8.5 g cornmeal agar	0.5 g yeast
   3.5 g glucose	extract (Brewer's)
   5 g sucrose	0.05 g KH2PO4
   500 ml distilled water

   Mix the dry ingredients into the water while stirring and heat the solution until the agar is completely dissolved. Pour into two 500 ml flasks and seal each flask with foil. Autoclave for 15 minutes at 15 psi.

2. Petri plate preparation:

   Cool the sterile crossing agar until the flask is easy to handle. Under a sterile hood, pour sterile plates 1/2 to 2/3 full. After the agar has set, tape five plates together and invert to store. It is best to pour the plates at least a day in advance of inoculation. Examine the plates just before inoculating and discard any that appear contaminated. (One liter of agar yields approximately 35-40 petri plates.) One plate per lab yields sufficient material, but students can be given individual plates if so desired. We prepare two plates per lab, one "young'' and one "old'' as described below.

1. The following procedures should be carried out in a hood using sterile technique;
   1. a. With a sterile spatula (dip in alcohol and flame), section the stock culture plates of Sordaria into 1/4 inch squares.
   2. b. Transfer one square of the wild strain to a location one-third along a diameter of a petri plate containing crossing agar. The upper surface of the square with Sordaria on it should be placed downward on the fresh agar. Inoculate half the plates needed for the lab exercise in this manner. Using the same procedure, place a square of the mutant strain one-third along the same diameter opposite the wild strain.

2. Place the inoculated plates right side up in an incubator with a pan of water at the bottom. Light is not necessary, but temperature should be 22-24 ° C.

Inoculate the remaining plates in the described manner and incubate. This will provide some ``younger'' plates to insure mature asci in seven to ten days.

Two to Three Days before Lab

Plates should be checked at seven days incubation for mature asci along the mating area. If they are mature (black perithecium as seen under dissecting microscope), store the inverted plates in the refrigerator until the lab period.

Morning of Lab

1. Check all plates for areas of crossing-over by mounting and crushing perithecia as described in the laboratory manual. The area that best shows crossing-over is along the midline of the plate between the two strains. Mark the bottom of each plate where perithecia with crossovers are observed.

2. Select a male and female preserved Ascaris. (The female is larger and longer than the male.) Rinse with water and dissect so students can view the reproductive system anatomy. Pour water or preserving fluid over specimen. Cover the dissection pan with plastic wrap or clear Plexiglas. This preparation helps students understand how the slide of Ascaris eggs was made.

NOTES

1. On occasion, other organisms can grow along with the Sordaria. Although this does not seem to interfere with the mating, the foreign growth should be removed before giving the cultures to the students.

2. Agar plates can be poured successfully on a prepared bench top, however, a transfer hood reduces contamination. A simple hood can be constructed from a 36" x 48" x 3/8" piece of Plexiglas. Cut two sides, 17" x 11 1/2" x 19" (quadrilateral shaped), one top, 24" x 18", and a back, 24" x 18 1/2". Construct a three-sided box with a sloped top. Prior to pouring the plates or making culture transfers, swab the entire inside surfaces with an antiseptic, such as alcohol or bleach.

CLASSROOM SUGGESTIONS

1. Students have a difficulty interpreting the Ascaris slides. Be prepared to explain.

2. A dissecting scope helps to see the perithecia when students are taking samples. In skilled hands, the tip of a probe can be used to remove a few of the perithecia. However, a flat toothpick scraped gently across the agar surface can provide similar results.

3. Damage to the mature asci can be minimized by observing their release under LOW power of the compound microscope. With a blunt pencil or stick, gently press the coverslip until the star-shaped cluster of asci are free of the perithecium. By drawing a drop of water through the pressed preparation, the asci can be freed enough to make an accurate count. The students can practice this technique by first selecting either a tan or black perithecium at the edge of the culture.

4. Results for crossing-over should fall in the 25--30% range. Preparation of a slide for viewing the crossing-over is quite simple. However, students might not be too successful on the first or second attempts. Most often they press too hard and the asci rupture.

5. Students should be encouraged to share successful slide preparations with each other. The laboratory can be completed in two hours but depends on how long the students need for the microscope studies.

6. Minimum homework would be to prepare a histogram of the percentage of crossing-over observed by students in the class.

ANSWERS TO CRITICAL THINKING QUESTIONS

1. Critical value tables are based on probabilities of random events. If each researcher chose an acceptable level of variation and did not use standard critical value tables, there would be tendency for some researchers to argue for large variation as being acceptable. Standard tables and conventions of science prevent this from happening.

2. Diploid numbers represent chromosomes in pairs. Pairs will give an even number.

SUPPLEMENTAL MATERIALS

Bio Sci II, videodisc--contains sequences on meiosis. Dubuque, IA: Wm. C. Brown Publishers. See appendix.

Genetics of Sordaria, film loop. Concord, MA: KALMIA Corp. #56-1610

Mitosis and Meiosis, color film. Chicago, IL: Encyclopaedia Britannica Educational Corp.

Sordaria, 3 minute film. Ames, IA: Media Resources Film Library, Iowa State University. #54902