Colchicine and the Onion Root Tip Mitotic Index
During a practical, a student germinates onion bulbs and collects root tips 1 mm long. After fixing in ethanol–acetic acid, hydrolysing in 1 M HCl and staining with acetocarmine, she squashes each tip and counts 400 cells from five random fields at ×400. In the untreated root she records: interphase 320, prophase 40, metaphase 24, anaphase 12, telophase 4. A second root is soaked in 0.05% colchicine for 3 hours before fixation; its counts are interphase 307, prophase 30, metaphase 60, anaphase 2, telophase 1. A classmate concludes that colchicine stimulates cell division because the treated root has more metaphase cells. The teacher asks the student to calculate mitotic indices, examine the phase distribution and decide whether the conclusion is sound. The student is also asked why only the very tip is used and why five fields rather than one are counted.
1. Calculate the mitotic index for each root and compare the phase distributions. Does the data show that colchicine stimulates cell division?
Read the worked answer
Mitotic index = dividing cells ÷ total cells × 100.
Control: (40 + 24 + 12 + 4) ÷ 400 = 80 ÷ 400 = 0.20 = 20.0%. Treated: (30 + 60 + 2 + 1) ÷ 400 = 93 ÷ 400 = 0.2325 = 23.3%.
The indices are close: a difference of 3.3 percentage points, not a doubling of division. The phase distribution changes much more sharply. In the control, metaphase is 24 ÷ 80 = 30.0% of dividing cells; in the treated root it is 60 ÷ 93 = 64.5%. Anaphase plus telophase falls from 16 ÷ 80 = 20.0% to 3 ÷ 93 = 3.2%. This is a shift into metaphase, not a proportional rise in all phases. Colchicine inhibits microtubule assembly, so the spindle cannot pull chromatids to the poles and cells accumulate in metaphase. The slightly higher mitotic index is explained by arrest, not by stimulation of entry into mitosis. A true mitogen would raise prophase, metaphase, anaphase and telophase together. The classmate's conclusion is therefore not supported.
2. Why is only the 1 mm root tip used, and why count five random fields rather than one? What further replication would strengthen the conclusion?
Read the worked answer
The root apical meristem lies in the terminal 1 mm. Cells there are actively cycling, so prophase, metaphase, anaphase and telophase are frequent enough to count. Older tissue behind the tip contains differentiated cells, mostly in interphase or G0, and including it would dilute the mitotic index and blur treatment differences.
Five random fields reduce sampling bias. One field might contain only interphase cells or a local clump of dividing cells. Counting 400 cells across five fields gives a more stable proportion. To strengthen the conclusion, the student should use several roots per treatment as biological replicates, prepare several slides, and count blind so that the observer does not know which treatment is being scored. She should also fix all roots at the same time after treatment, because mitotic index varies with time of day and with duration of colchicine exposure. Means and ranges should be reported, not a single root per treatment.
3. The treated root has only 2 anaphase and 1 telophase cell. Explain why these few cells are not evidence that colchicine accelerates anaphase.
Read the worked answer
If colchicine blocked spindle formation, anaphase and telophase should become rare, because chromatids cannot be separated and moved to opposite poles. That is what the data show: anaphase plus telophase is 3 out of 93 dividing cells, compared with 16 out of 80 in the control. A drug that accelerated anaphase would increase late stages, not reduce them.
The few late-stage cells can be explained in several ways: they may have entered anaphase before the colchicine concentration became effective; they may be escapes from the block; or they may come from a less exposed part of the tip. Their low number is consistent with metaphase arrest, not acceleration. A 3-hour treatment is short enough that some cells could still complete division. The overall pattern, especially the rise in metaphase and fall in anaphase plus telophase, supports the conclusion that colchicine arrests cells in metaphase.