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STUDENT OBJECTIVE

Stages of mitosis are observed in cells of animal (whitefish blastula) and plant (sectioned onion root). Students stain onion root tips and count the number of chromosomes in onion cells. Students make histograms of the observed number of chromosomes per cell from class data and calculate an average and standard deviation.

EQUIPMENT	AMOUNT
	(Class of 24 with 8 groups)
Compound microscopes Water bath 60oC	1/student 1/lab
MATERIALS
Prepared slides (sharing possible to reduce costs) Whitefish blastula (CBS#30-8946)* Onion root tip sectioned (CBS#30-2396)* Lens tissue, 5 cm x 7 cm cut from pkg Onion root tips two to four days old Glass pestles, glass rod 4" x 1/8"; fire polish the end; flatten by filing Vials, screw-cap, 15 ml to 20 ml capacity Razor blades Forceps Watch glass, small Slides Coverslips, #1 medium square glass Tissue, blotting Glass etching marker (optional for labeling permanent slide) Dry ice (optional) Container for disposal of razor blades and coverslips	24/lab 24/lab 8 pkg/lab 12 onions/lab 12/lab 24/lab 12/lab 12/lab 12/lab 24/lab 1/2 oz/lab 2 boxes/lab 1/lab 1/2 pound/lab 1/lab

Please refer to the Appendix for name and address of supplier.

SOLUTIONS

Glacial acetic acid-methanol fixative (1:3)
1 M hydrochloric acid (HCl)
45% acetic acid (CH₃COOH)
Feulgen stain

PREPARATION

Three Weeks before Lab

1. Depending on the time of the year, onion sets can be purchased in local garden centers. If not available, order by phone from a reliable garden supply house. Yellow onion sets have the best "keeping" quality. If bulbs are not to be used immediately, store in a cool place (10—15^oC). Onions from a grocery must be started earlier than sets. You might also try a green onion but be sure to clip off old roots.
2. Upon receipt of onions, sprout four to six bulbs to check the time of most vigorous growth. Pour 1" vermiculite into a small flat container and thoroughly soak with tepid water. Drain off excess and push bulbs approximately 1/2" into the wet medium. Set in a warm well-lighted area (22—24^oC) and check each day for growth; add water so that vermiculite stays moist but not wet.

Week before Lab

1. Prepare sprouting trays and plant onions according to the timetable established. Two to four days’ sprouting will provide numerous root tips in cell division. Mitotic cell division in roots is no longer at the maximum when green tops begin to show.
2. Hydrochloric acid preparation:

   1 M HCl 83 ml commercial concentrated HCl/distilled water to make 1 liter

   In a fume hood, add the acid to the water while stirring constantly. Wear goggles! Be aware of heat of solution.

   Store in a glass bottle. Shelf life: indefinite

3. 45% acetic acid preparation: 270/ ml glacial acetic acid/330 ml distilled water

   Prepare as in #2. Shelf life: indefinite

4. Feulgen stain preparation:

Day of Lab

1. All work areas should be covered with several thicknesses of newspaper. Feulgen stain stains the skin, clothes, and work areas. Household bleach can remove the stains from most surfaces.
2. Fixative preparation:

NOTES

1. Onion sets are not available in all areas year round. You may pre-fix roots as in #2 or order root tips from a supply house when onion sets are not in season. Alternatively, small whole onions from the grocery store may be used.
2. Previously fixed root tips can also be used to begin this exercise. Harvest the tips when the majority are 5 mm in length and immerse in the alcohol-acetic acid fixative for 24 hours. Transfer the tips to a 70% ethanol solution for indefinite storage in the refrigerator. Students should thoroughly rinse the tips prior to proceeding with the hydrolysis.

CLASSROOM SUGGESTIONS

1. Tell students to cut roots off onion at the base of the root. Put root on slide and remove 2—3 mm of the tip for staining and discard remainder. This insures that all roots remaining on stock onions have tips. Otherwise, students may get roots that have already had tips removed.
2. Limiting the number of times the water bath is opened during the hydrolysis will help to maintain a constant temperature.
3. During the staining procedure, students may find it difficult to decant the liquids without losing the root tips. Easy transfer of the tips with a forceps is achieved by pouring the entire contents into a small watch glass.
4. This lab gives successful and satisfying results for most students. Three areas of difficulty are: not pulverizing the tissue long enough, not exerting enough firm, downward pressure during the squash, and not focusing the microscope up and down through the many planes of the preparation. Students can usually complete the laboratory in 2—2 1/2 hours.
5. A minimum homework assignment would be to pool class data on the number of chromosomes in onion cells and have students plot a histogram and calculate an average and standard deviation.
6. An inexpensive, but excellent animated meiosis simulator for the Macintosh is available from Intellimation (phone 800-346-8355). Ask about Mitosis and Meiosis (553-A) Site licenses are available.
7. Check out the links for this lab topic at http://auth.mhhe.com/biosci/genbio/dolphin/ You will find useful materials for developing your lab introduction or summary, and in some cases, you may want to tell students to connect to a particular site for further information.

ANSWERS TO CRITICAL THINKING QUESTIONS

1. Selection for chromosome number occurs because of physical confines of space in the nucleus and during cell division.
2. No. There are several million different species, but chromosome number is usually below 100. Obviously several species have the same number of chromosomes.
3. Before mitosis each chromosome duplicates itself. Mitosis distributes one duplicate to each daughter cell. Therefore, they must be identical.

SUPPLEMENTAL MATERIALS