In cell division observation experiments, actively dividing tissues are stained and observed with an optical microscope to identify cells in interphase, prophase, metaphase, anaphase, and telophase.
Typical specimens include the root tip meristems of onions and green onions. Near the root tip, new cells required for plant growth are actively produced, making it easier to find cells at various stages of division.
During mitosis, replicated chromosomes are distributed equally to two daughter cells. The division stage of each cell is identified from features such as chromosome condensation, alignment at the equatorial plane, separation of sister chromatids, and formation of two nuclei.
This article assumes an experiment observing a squash preparation of an onion root tip and summarizes the introduction, precautions, result items, reference experimental values, calculations, sources of error, example results, and example discussion.
Note:
This article is a study reference for discussing results obtained in biology experiments at schools, universities, and similar settings. The experimental conditions and measurements shown are examples for explanation and are not actual observation results.
When using fixatives, dissociation solutions, or stains, avoid contact with skin, eyes, and clothing, and follow the instructor’s or laboratory manual’s instructions regarding ventilation and protective equipment.
When squashing slide and cover glasses, take care to avoid cuts from broken glass. When using a high-power microscope objective, do not allow the lens tip to contact the slide preparation.
Stages of Mitosis
Interphase
The nuclear membrane and nucleolus are visible, while the chromosomes are spread out as threadlike chromatin and cannot be clearly distinguished individually. DNA replication and cell growth proceed during this stage.
Prophase
Chromosomes condense, becoming thicker and shorter and easier to distinguish under the microscope. The nuclear membrane and nucleolus gradually become indistinct.
Metaphase
Chromosomes align near the equatorial plane at the center of the cell. This is a stage in which chromosome shape and number are relatively easy to observe.
Anaphase
Sister chromatids separate and move toward opposite poles of the cell. They are observed as two groups of chromosomes moving apart from each other.
Telophase
Nuclear membranes reform around the chromosomes that have moved to opposite poles, and the chromosomes gradually decondense. In plant cells, a cell plate forms at the center of the cell.
Main Items to Record in the Results
- Specimen observed
- Observation site
- Fixation, dissociation, and staining conditions
- Eyepiece magnification
- Objective magnification
- Total magnification
- Number of fields observed
- Total number of cells
- Number of interphase cells
- Number of prophase cells
- Number of metaphase cells
- Number of anaphase cells
- Number of telophase cells
- Number of dividing cells
- Mitotic index
- Proportion of each mitotic phase
- Appearance of chromosomes
- Presence or absence of a cell plate
- Presence or absence of abnormal division
- Sketches or images of representative cells
Reference Experimental Conditions
| Item | Reference Condition |
|---|---|
| Specimen | Root tips of sprouted onions |
| Observation site | Meristem approximately 1–2 mm from the root tip |
| Preparation | Squash preparation |
| Stain | Aceto-orcein or acetocarmine |
| Eyepiece | 10× |
| Objectives | 10×, 40× |
| Main observation magnifications | 100×, 400× |
| Number of fields observed | 5 fields |
| Total cells observed | 500 cells |
Reference Experimental Values
Number of Cells at Each Stage
| Cell-cycle Stage | Number of Cells | Percentage of All Cells |
|---|---|---|
| Interphase | 410 | 82.0% |
| Prophase | 48 | 9.6% |
| Metaphase | 18 | 3.6% |
| Anaphase | 10 | 2.0% |
| Telophase | 14 | 2.8% |
| Total | 500 | 100% |
Number of Dividing Cells in Each Field
| Field | Total Cells | Dividing Cells | Mitotic Index |
|---|---|---|---|
| 1 | 96 | 16 | 16.7% |
| 2 | 102 | 20 | 19.6% |
| 3 | 98 | 17 | 17.3% |
| 4 | 101 | 18 | 17.8% |
| 5 | 103 | 19 | 18.4% |
| Total | 500 | 90 | 18.0% |
Characteristics of Observed Images at Each Stage
| Stage | Reference Observation |
|---|---|
| Interphase | The nucleus was clear, and individual chromosomes could not be distinguished. |
| Prophase | Condensed chromosomes appeared threadlike or clumped within the nucleus. |
| Metaphase | Chromosomes appeared aligned near the center of the cell. |
| Anaphase | Chromosomes had separated into two groups and were moving toward opposite poles. |
| Telophase | Two nuclei had formed, and a cell plate was visible in the center. |
Example Comparison of Treatment and Control Groups
| Condition | Total Cells | Dividing Cells | Mitotic Index |
|---|---|---|---|
| Control | 500 | 90 | 18.0% |
| Low-temperature treatment | 500 | 62 | 12.4% |
Calculation Methods
Number of Dividing Cells
Number of dividing cells = Prophase + Metaphase + Anaphase + Telophase
= 48 + 18 + 10 + 14
= 90 cells
Mitotic Index
The mitotic index is the percentage of all observed cells that are in mitosis.
Mitotic index (%) = Number of dividing cells ÷ Total number of cells × 100
= 90 ÷ 500 × 100
= 18.0%
Percentage of Each Mitotic Phase
The percentage of prophase cells is calculated as follows.
Prophase percentage (%) = Number of prophase cells ÷ Total number of cells × 100
= 48 ÷ 500 × 100
= 9.6%
Percentage of Each Stage Within Mitosis
If 48 of the 90 dividing cells were in prophase, the calculation is as follows.
Prophase percentage within mitosis (%) = 48 ÷ 90 × 100
≈ 53.3%
Estimating the Duration of Each Stage
Assume that the cell population is in a steady state and that the percentage of cells at each stage is proportional to the time spent in that stage.
If the entire cell cycle is assumed to be 24 hours, the estimated duration of prophase is calculated as follows.
Estimated prophase duration = Number of prophase cells ÷ Total number of cells × Cell-cycle duration
= 48 ÷ 500 × 24 hours
= 2.304 hours
≈ 2 hours 18 minutes
The estimated durations of the stages are as follows.
| Stage | Percentage | Estimated Duration Assuming a 24-hour Cycle |
|---|---|---|
| Interphase | 82.0% | 19.68 hours |
| Prophase | 9.6% | 2.304 hours |
| Metaphase | 3.6% | 0.864 hours |
| Anaphase | 2.0% | 0.480 hours |
| Telophase | 2.8% | 0.672 hours |
Rate of Change in Mitotic Index Due to Treatment
For a control value of 18.0% and a low-temperature treatment value of 12.4%, the calculation is as follows.
Rate of change (%) = (Treatment − Control) ÷ Control × 100
= (12.4 − 18.0) ÷ 18.0 × 100
≈ −31.1%
Thus, the mitotic index in the low-temperature treatment group can be described as approximately 31% lower than that in the control group.
Main Sources of Error
| Source of Error | Effect on Observation | Likely Effect on Results | Improvement |
|---|---|---|---|
| Using tissue away from the root tip | Fewer dividing cells | Underestimates mitotic index | Select meristem immediately adjacent to the root tip |
| Specimen too thick | Cells overlap and chromosomes are difficult to see | Increases errors in stage identification | Squash sufficiently to make the preparation thin |
| Insufficient squashing | Cells remain crowded | Underestimates total cell count | Press evenly without shifting the cover glass |
| Excessive squashing | Cells or chromosomes are damaged | Increases abnormal images | Avoid excessive force |
| Insufficient staining | Low chromosome contrast | Dividing cells may be missed | Standardize staining time and temperature |
| Excessive staining | The entire cell becomes dark | Chromosomes are difficult to distinguish from one another | Remove excess stain |
| Poor fixation | Cell structures deteriorate | Chromosome shapes become unclear | Standardize fixation conditions |
| Insufficient dissociation | Cells are difficult to separate | More overlap | Use appropriate dissociation conditions |
| Excessive dissociation | Cells are damaged | Cell outlines and chromosomes deteriorate | Observe the specified treatment time |
| Incorrect focus | Chromosome images are blurred | Stages may be misidentified | Adjust focus with the fine-focus knob |
| Confusing prophase with interphase | Early-condensing cells are misclassified | Biases prophase or interphase percentages | Standardize classification criteria beforehand |
| Confusing anaphase with telophase | Degree of nuclear membrane reformation is misjudged | Changes anaphase/telophase percentages | Use both the cell plate and nuclear membrane for classification |
| Counting the same cell twice | Total cell count increases | Percentages become inaccurate | Move the field systematically in one direction |
| Biased field selection | Only areas rich in dividing cells are selected | Overestimates mitotic index | Select fields randomly or at fixed intervals |
| Too few cells observed | Random bias becomes large | Stage percentages become unstable | Count many cells across multiple fields |
| Differences in specimen growth condition | Mitotic activity differs | Mitotic index varies among individuals | Standardize root length and culture conditions |
| Different sampling times | Subject to daily variation in mitotic activity | Results vary by experiment date and time | Collect specimens at the same time |
Why Interphase Cells Are the Most Numerous
When an observed cell population is in a steady state, the number of cells observed at each stage is approximately proportional to the time spent in that stage.
Interphase occupies more time than the individual stages of mitosis because DNA replication, cell growth, and preparation for division occur during interphase. Therefore, most observed cells are in interphase.
Why Prophase Cells Are the Most Numerous During Mitosis
During prophase, many changes occur, including chromosome condensation, nuclear membrane breakdown, and spindle formation. Because prophase often takes longer than metaphase, anaphase, or telophase, more prophase cells are observed among dividing cells.
Why Chromosomes Are Easy to Observe in Metaphase
In metaphase, chromosomes are highly condensed and align near the equatorial plane at the center of the cell. Their positions and shapes are therefore relatively easy to distinguish.
Why a Cell Plate Forms in Plant Cells
Plant cells have rigid cell walls, so they cannot divide by pinching inward from the outside as animal cells do.
Instead, a cell plate forms at the center of the cell and expands outward to become a new cell wall separating the two daughter cells.
Example of How to Write the Results
A squash preparation of an onion root tip was prepared, and mitosis was observed using an optical microscope.
A total of 500 cells were counted across five fields: 410 in interphase, 48 in prophase, 18 in metaphase, 10 in anaphase, and 14 in telophase.
There were 90 dividing cells in total, giving a mitotic index of 18.0%.
Among mitotic cells, prophase was the most common stage. In metaphase, chromosomes were aligned at the center of the cell; in anaphase, chromosomes separated into two groups and moved toward opposite poles. In telophase, two nuclei and a cell plate were observed.
Points to Connect the Results to the Discussion
- What difference was observed between the numbers of interphase and mitotic cells?
- What does the mitotic index indicate?
- Which stage was most common among dividing cells?
- Can the relationship between the proportion of cells at each stage and the duration of that stage be explained?
- What criteria were used to identify prophase, metaphase, anaphase, and telophase?
- Why were metaphase chromosomes easy to see?
- How does cytokinesis in plant cells differ from that in animal cells?
- How would the mitotic index change if tissue other than the root tip were observed?
- How did specimen thickness and staining affect classification?
- Was there any bias in field selection?
- Was the number of observed cells sufficient?
- If the mitotic index differed between treatment and control groups, what might explain the difference?
Discussion Example
In this experiment, meristematic tissue from an onion root tip was observed, and the numbers of cells at each stage of the cell cycle were counted.
Of the 500 cells observed, 410 were in interphase, accounting for 82.0% of the total. In contrast, 90 cells were undergoing mitosis, giving a mitotic index of 18.0%.
Interphase cells were the most numerous because interphase occupies most of the cell cycle. DNA replication, cell growth, protein synthesis, and preparation for division occur during interphase, so it requires more time than the mitotic stages from prophase through telophase.
Assuming that the proportion of cells at each stage is proportional to the duration of that stage, a 24-hour cell cycle would include approximately 19.7 hours of interphase and approximately 2.3 hours of prophase.
However, this estimate assumes that the observed cell population is in a steady state and that all cells divide with the same cycle. In reality, differences among individual cell cycles and the presence of cells that have stopped dividing may affect the estimate.
Prophase was the most common mitotic stage. This may be because many changes occur during prophase, including chromosome condensation, nuclear membrane breakdown, and spindle formation, making it longer than metaphase or anaphase.
In metaphase, condensed chromosomes were observed aligned near the equatorial plane at the center of the cell. Because chromosomes are highly condensed at this stage, their shapes and positions could be observed relatively clearly.
In anaphase, sister chromatids separated and two groups of chromosomes moved toward opposite poles. This process distributes replicated genetic information equally to the two daughter cells.
In telophase, two nuclei formed at opposite poles, and a cell plate was observed at the center of the cell. Because plant cells have rigid cell walls, cytokinesis occurs by forming a cell plate from the center rather than by constriction as in animal cells.
The mitotic index is an indicator of the level of cell division activity in the observed tissue. A mitotic index of 18.0% suggests that many cells in the observed root-tip tissue were proliferating.
Because new cells required for plant elongation are produced at the root tip, the mitotic index is expected to be higher there than in mature regions farther up the root.
One source of error is the difficulty of distinguishing early prophase from interphase. In cells where chromosome condensation had only just begun, the chromosomes may not yet have been clear, and such cells may have been counted as interphase.
There is also ambiguity between late anaphase and early telophase. Even when the two chromosome groups have reached the poles, classification may differ among observers if the nuclear membrane or cell plate is not yet clear.
In thick parts of the specimen, cells overlapped and chromosome images became unclear, so dividing cells may have been overlooked. This would cause the mitotic index to be underestimated.
Conversely, intentionally selecting fields in which many dividing cells are visible would overestimate the mitotic index. Therefore, fields should be selected randomly or at fixed intervals, and the same cells should not be counted more than once.
Observation accuracy can be improved by using the same region of the root tip, squashing the specimen thinly and uniformly, standardizing fixation, dissociation, and staining conditions, and confirming classification criteria among multiple observers.
Increasing the number of observed cells and preparing specimens from multiple root tips or individuals can also reduce bias arising from a single field or specimen.
Overall, interphase cells were the most numerous in the onion root tip, while prophase was the most common mitotic stage. The sequence of chromosome condensation, alignment, separation, and nuclear reformation also demonstrated how mitosis distributes genetic information equally to two daughter cells.
Discussion Example When the Mitotic Index Was Low
A lower-than-reference mitotic index may have resulted from observing mature tissue farther from the root tip, missing dividing cells because of insufficient staining, cell overlap in a thick specimen, or suppression of cell division by low temperature or nutrient deficiency.
Discussion Example When the Mitotic Index Was High
A higher-than-reference mitotic index may have resulted from preferentially selecting fields rich in dividing cells, counting interphase cells as prophase, or observing a particularly mitotically active region within the root tip.
Discussion Example When Many Cells Were in Metaphase
If many cells were in metaphase, chromosome movement or spindle function may have been inhibited, causing cells to arrest near metaphase. However, classification error in which late prophase cells were identified as metaphase should also be considered.
Discussion Example When It Was Difficult to Identify Each Stage
Difficulty identifying each stage may result from the continuous nature of cell division, with no sharp boundaries between stages, as well as insufficient staining, incorrect focus, or overlapping cells. Classification accuracy can be improved by using multiple criteria, including chromosome condensation, chromosome position, the nuclear membrane, and the cell plate.
Summary
In cell division observations, meristematic tissue such as root tips is used to identify cells in interphase, prophase, metaphase, anaphase, and telophase.
Calculating the mitotic index and the percentage of cells at each stage from observed cell counts makes it possible to estimate the level of cell division activity in a tissue and the relative time spent in each stage.
Interphase cells are generally the most numerous, while prophase cells are commonly the most frequently observed among mitotic cells.
In the discussion, it is important to relate factors such as root-tip sampling position, staining, specimen thickness, stage classification, field selection, and number of observed cells to deviations in the mitotic index and the proportions of each stage.
