Chromosomes were first seen by C. Nägeli in 1842, and named in 1888 by W. Waldeyer. Walther Flemming studied and documented the behavior of chromosomes during cell division, a process he termed mitosis. We will perform experiments similar to these early scientists.

Cell division is especially rapid in the growing root tips of sprouting seeds. The chromosomes in dividing root tip cells can be demonstrated if, after we sprouting seeds or bulbs,  we harvest, fix acid digest, stain and squash, and view under a microscope the root tips. The chromosomes viewed in our lab, best to least, have been: onion, wheat, lentils, barley, rye and alfalfa.

SUPPLIES:
 

1. Carnoy's fixative (1:3 HOAc + EtOH)
2. 1N HCl
3. Feulgen stain
4. 45% HOAc
5. Freshly sprouted seeds (rye, wheat, lentils, alfalfa, onion, etc), about 2-3 cm long.

EQUIPMENT:
 

1. Wasserman tubes (13 x 100 mm) with corks
2. Pasteur pipets
3. Constant temperature "hot block", 60^oC
4. microscope slides
5. razor blade
6. cover slips

THE DAY PREVIOUS:
 

1. Label tube with sprout type, seat number, initials and the date.
2. Cut off the last 6 mm (1/4th inch) of root tip from freshly sprouted seeds, immerse in 2 mL Carnoy's fixative.
3. Fix the root tips for at least 24 hours in Carnoy's fixative.

DIGEST, STAIN AND SQUASH THE FIXED ROOT TIPS:

2. Suck up two fixed root tips with a Pasteur pipet, transfer to an empty Wasserman tube, labeled with your seat number and the type of root tip. Draw off xs fluid.

3. Digest with HCl: Add 1-2 mL of 1N HCl with a Pasteur pipet, incubate at 60C for 12 minutes in a constant temperature "hot block."

4. Stain with Feulgen stain: Remove HCl with Pasteur pipet, discard in running cold water. Add 1 mL Feulgen stain (caution: this stain does not look brightly colored, but stains strongly. Do not get on clothes, fingers, books, etc.) Let sit at R.T. for 10 minutes until the very tip of the root shows distinct dark coloring.

5. Prepare the squash: Put a drop of 45% HOAc on a clean microscope slide. Remove the Feulgen stain with a Pasteur pipet, discard into the drain. Place the soft root tip in the HOAc on the slide. With a scalpel or razor blade, remove all but the red-stained very tip of the root. Cover with a cover slip, place on white paper and tap gently with the eraser of a pencil until the stained tip is spread out to a faint purple monolayer. Do not smear side-wise, it shears the chromosomes.

6. Examine under the microscope at low power to ensure that the cells are adequately spread to a monolayer. If so, examine under higher power. Locate mitotic figures (near the tip end), and switch to oil immersion (1000x).

7. Illustrate examples of each mitotic stage (pro-, meta-, ana- and telophase). Prepare a second squash with a different species, and illustrate its mitotic stages, noting any differences observed between the two species.

8. Show chromosomes of two species of plants to the instructor for 5 points each.