Exercise 7 - Mitosis and Chromosomes

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	1/student
Water bath 60 ° C	1/lab

MATERIALS

SOLUTIONS

Glacial acetic acid-methanol fixative (1:3)
1 M hydrochloric acid (HCl)
45% acetic acie (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°C). Onion root tips can also be taken from onion seed germinated on filter paper in petri dishes.

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°C) and check each day for growth; add water so that vermiculite stays moist but not wet.

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:

   Following the same procedure as #2, make 0.15 M HCl by adding 6.3 ml concentrated HCl to distilled water to make 500 ml.

   Then, add 5 g leucobasic fuchsin (Fuchsin Basic) and 9.5 g sodium meta-bisulfite to 500 ml 0.15 M HCl. Stir two hours. Add 1 g activated charcoal; shake two minutes.

   Filter solution using a Buchner funnel with Whatman #1 filter paper. Make certain a trap is in line while filtering. The filtrate should be clear. If yellow, add additional activated charcoal, refilter.

   Store in a brown bottle in a refrigerator. Shelf life: six months. Prior to using, dilute by adding two parts of stock solution to one part water. For class use, pour diluted solution into brown dropper bottles.

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:

   Make fresh daily. 80 ml glacial acetic acid/240 ml 100% methanol

   Wear protective goggles and clothing when mixing fixative.

NOTES

1. Some garden suppliers will treat the bulbs with a growth inhibitor to obtain longer storage of onion sets. If only treated bulbs are available, onion seeds can be used to obtain the root tips. Sprinkle the seeds in a petri dish on wet paper toweling and cover with moist paper toweling. Set in a warm place (22-24°C) and check daily. Add water to maintain the moisture content. In approximately seven days, adequate root tips for the exercise will be available.

2. The fastest and most reliable source of roots is onion sets. Yellow and white onions purchased in the grocery store will also yield roots. Remove all dry skin and old roots and insert bottom third of bulb in moist vermiculite.

3. 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. Prewarming the HCl while the tips are in the fixative will also help to assure the proper time of processing.

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 thro ugh 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. Repeat new entry from page 32 here as well as on 32.

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.

SUPPLEMENTAL MATERIALS

Bio Sci II, videodisc--contains sequences on mitosis in plants and animals. Dubuque, IA: Wm. C. Brown Publishers. See appendix.
Models of mitosis' stages in Ascaris (CBS#56-1650)
Onion Mitosis Set, 35 mm slides (CBS#48-1135) Carolina Biological Supply, Burlington, NC.