What you need to know
- 3.15Prepare and stain a root tip squash to observe mitotic stages.
- 3.16Calculate mitotic indices.
- 3.14Relate observations to the cell cycle and mitosisNuclear division that produces two genetically identical nuclei with the same number of chromosomes as the parent..
By the end, you should be able to…
- Explain preparation steps and risk controls.
- Recognise mitotic stages using visible evidence.
- Calculate indices and evaluate sampling and timing assumptions.
Build your understanding
Choose the actively dividing region
Use a teacher-selected growing root, such as onion or garlic. The meristem just behind the root cap contains many dividing cells. Sampling a mature region further from the tip changes the cell population and can lower the observed proportion in mitosisNuclear division that produces two genetically identical nuclei with the same number of chromosomes as the parent..
This guide prepares you for supervised laboratory work. Use your school’s approved protocol, reagent concentrations, heating conditions and waste procedure. Prepared slides can support recognition practice, but do not replace learning how to prepare and stain the squash.
Prepare a thin, stained layer
Under the approved protocol, acid treatment and controlled warming soften tissue by disrupting the material joining neighbouring cells, helping them separate. Rinse as directed before subsequent staining. Excess treatment can damage the preparation.
A suitable stain, such as acetic orcein in an approved method, increases chromosomeA long DNA molecule wound around histone proteins. After replication it is two sister chromatids joined at a centromere. contrast; fixation preserves a snapshot. Transfer the small meristem region to a slide, add the specified liquid and gently tease it apart. Lower a coverslip and apply controlled vertical pressure through protective paper as instructed. Do not twist or slide the coverslip.
Squashing spreads the cells into a thin layer, reducing overlap and allowing light through. Too little pressure leaves overlapping cells; excessive pressure can damage cells or break glass. Start at low magnificationHow many times larger an image is than the real object: magnification = image size ÷ actual size., locate the meristem and then use a higher objective with fine focus.
Risk controls have a reason
Wear eye protection. Acid and acidic stain can damage skin and eyes; avoid contact and vapour, keep containers managed as directed, and report spills immediately to the teacher. Follow the school response procedure rather than cleaning an unknown spill yourself.
Hot water baths can scald, so use the prescribed handling tools. Cutting implements and broken slides can injure: cut on a suitable surface away from fingers, use controlled pressure and place damaged glass in the designated container. Keep liquids away from electrical equipment. Do not direct sunlight into a microscope mirror.
Identify and record evidence
Record interphaseThe stage of the cell cycle between divisions, when the cell grows and its DNA replicates. and each mitotic stage in a tally table. prophaseThe first stage of mitosis: chromosomes condense and the nuclear envelope breaks down. shows condensing chromosomes; metaphaseThe stage of mitosis when chromosomes line up at the equator, attached to spindle fibres by their centromeres. an equatorial arrangement; anaphaseThe stage of mitosis when centromeres divide and sister chromatids are pulled to opposite poles. two separating groups; telophaseThe last stage of mitosis: chromosomes reach the poles and a nuclear envelope forms around each set. reforming nuclei. Draw only what is visible, with clear outlines, labels and stated magnification or a calibrated scale.
Choose a consistent cell-boundary rule so cells on adjacent fields are not counted twice. Sample fields by a preselected systematic or random approach within the defined meristem region, not by hunting for views rich in mitosis. Record ambiguous cells separately and explain how they are handled. Repeat across independently prepared roots.
Calculate and interpret
An illustrative count gives 156 interphase, 24 prophase, 10 metaphase, 4 anaphase and 6 telophase cells. Total = 200 and mitotic count = 44, so the index is 0.22, or 22%. Do not use only dividing cells as the denominator.
A phase’s fraction of all cells can estimate its fraction of cycle time only under assumptions: a representative, asynchronous population of actively cycling cells, broadly steady conditions and comparable cycles. It is an estimate, not direct timing. If the model cycle lasts 15 hours, 4/200 in anaphase suggests 0.30 hours = 18 minutes.
An arrest in a stage can increase its observed frequency. A higher mitotic index can therefore reflect slower completion, not faster tissue growth. Comparisons need controlled growth conditions, the same sampling region, independent roots and attention to preparation quality.
NOT: the index is not the number of cells you saw in metaphase alone, unless you defined it that way.
- Root tip is used because cells there divide
- Squash and stain so chromosomes can be seen
- State the total you counted
- Safety: follow the school’s stain and acid rules
Two traps this diagram blocks
- Do not count only the dividing cells and ignore the interphase cells in the denominator.
- Do not treat one field as the mitotic index of the whole root.
From the diagram alone
Question
A field contains 6 cells in mitosis and 44 other cells. Calculate the mitotic index and state one limitation. [3]
Show a model answer
The index is 6/50 = 0.12, or 12%. One field may not represent the root tip, so further fields counted with the same rule would make the estimate more reliable.
The root tip squash lab
Choose how to prepare a squash, classify cells field by field and calculate a mitotic index. The slide and counts are fictional.
Simulation only. Real squashes use acid and acidic stain: wear eye protection, avoid contact and vapour, use tools for hot baths and press the coverslip vertically through paper. Follow your school’s approved protocol.
Method 7 steps
- Cut the tip (about 5 mm) from a growing root, for example garlic or onion.
- Warm it in hydrochloric acid for the time your protocol gives, then rinse it in water.
- Put the tip on a slide, keep only the meristem just behind the root cap, and add a few drops of stain (for example acetic orcein or toluidine blue).
- Lower a coverslip, cover with paper and press down firmly and vertically to squash the tissue into a single layer, without twisting.
- Focus at low power, then high power, and find the meristem where cells are small and square with large nuclei.
- Score every cell in a field using a counting rule, then move systematically to further fields.
- Calculate the mitotic index, and estimate the time spent in each stage from the proportion of cells in it.
Aligned with the order of the specification practical. Simulation only: a teacher sets the real volumes, concentrations and risk controls; this is not a laboratory protocol.
Variables
- Independent
- None for a single root. In a comparison: for example root type, distance from the tip or treatment.
- Dependent
- Number of cells in each stage of mitosis, giving the mitotic index.
- Controlled
- Same preparation: acid time and temperature, stain, squash pressure
- Same region sampled (the meristem)
- Same counting rule and number of cells scored
Safety
- Hydrochloric acid and acidic stains are corrosive or irritant: wear eye protection and gloves, and avoid breathing vapour.
- Use tongs or forceps with warm acid or water baths. Take care with scalpels and press the coverslip through paper so it cannot cut you if it breaks.
- Follow your school’s risk assessment for the stain you use.
Follow your school’s risk assessment; these notes are not a substitute for it.
Interpreting
- Mitotic index = cells in mitosis ÷ total cells counted (including interphase). Most cells are in interphase because it is the longest part of the cycle.
- Time in a stage ≈ (cells in that stage ÷ total cells) × cell-cycle length.
- Results depend on preparation quality and on correctly identifying stages; record uncertain cells as unsure.
- Compare roots only when they were prepared and scored in the same way, and repeat with independent roots.
Guided method · step 1 of 8
- In 1 · Prepare the squash, just below these steps, set Tissue sampled to Meristem and Acid treatment (warm) to As protocol.
- Set Stain (e.g. acetic orcein) to Stained and Squash pressure to Firm, vertical.
- Press Prepare squash.
Mitosis happens in the meristem just behind the root cap. Warm acid breaks down the middle lamella holding cells together; a stain such as acetic orcein binds to DNA so chromosomes show up dark; firm, vertical pressure spreads the cells into a single layer.
1 · Prepare the squash
A fresh squash resets the tally.
Acid loosens the material holding cells together; stain increases chromosome contrast; squashing spreads cells into a thin layer.
Classify the selected cell
Use visible evidence only. Record overlapping, broken or unclear cells as unsure rather than guessing.
Running tally
Fields scored: 0 · key-checked: 0
Choose each preparation step, then prepare the squash.
2 · Calculate and interpret
| Stage | Count |
|---|---|
| Interphase | 0 |
| Prophase | 0 |
| Metaphase | 0 |
| Anaphase | 0 |
| Telophase | 0 |
| Unsure (excluded) | 0 |
| Scored total | 0 |
Evaluate the claim
Sample fields systematically within the meristem, score every countable cell and repeat with independently prepared roots.
Make your reasoning visible
Explain each preparation step
Acid helps cells separate; stain increases contrast; squash reduces overlap. These purposes are not interchangeable.
Include interphase in the total
Mitotic index counts cells in all mitotic stages over all scored cells.
State timing assumptions
A snapshot proportion estimates time only if the population and sampling support that inference.
Key explanations
- Staining makes chromosomeA long DNA molecule wound around histone proteins. After replication it is two sister chromatids joined at a centromere. easier to see.
- A cell with condensed, visible chromosomes is counted as being in mitosisNuclear division that produces two genetically identical nuclei with the same number of chromosomes as the parent..
- An interphaseThe stage of the cell cycle between divisions, when the cell grows and its DNA replicates. cell has a nucleus without visible separate chromosomes.
- Mitotic index is the number of cells in mitosis divided by the total number of cells counted.
- Counting several fields, with the same rule, makes the proportion less dependent on one view.
Avoid this misconception
The claim: The stain lets us watch the same living cell move through mitosis.
The correction: A fixed stained squash captures cells at different moments; it does not directly record their progression.
Check your understanding
Try each question before opening the suggested answer.
Check your understanding (continued)
Practice question
Retrieval and explanation · suggested answer
Why take cells close to the growing root tip?
Show suggested answer
The meristem contains actively dividing cells, increasing the chance of observing different mitotic stages.
Practice question
Retrieval and explanation · suggested answer
Why warm with acid and then squash under the approved method?
Show suggested answer
Acid treatment helps loosen tissue so cells separate; squashing spreads them into a thin layer with less overlap. Controlled treatment avoids excessive damage.
Practice question
Retrieval and explanation · suggested answer
A sample has 54 mitotic cells and 246 interphase cells. Calculate its mitotic index.
Show suggested answer
54/(54 + 246) = 0.18, or 18%.
Practice question
Retrieval and explanation · suggested answer
In a model steady population, 5% of cells are in metaphase and the cycle is 20 hours. Estimate metaphase duration and state a limitation.
Show suggested answer
0.05 × 20 = 1 hour. This assumes a representative asynchronous cycling population; stage arrest, non-cycling cells or biased sampling would undermine the estimate.
Practice question
Retrieval and explanation · suggested answer
Why is choosing the field with the most dividing cells a problem?
Show suggested answer
It biases the estimate upwards and does not represent the defined tissue region. Use a preselected sampling strategy and independent roots.
Take the idea further
Apply the same specification knowledge to an unfamiliar problem. Any extra assumptions are supplied in the question.
Stretch question
Root A has 10 mitotic cells out of 50; root B has 45 out of 150. Calculate the pooled index and the mean of the two root indices. Why do they differ?
Show a hint
One calculation weights roots by the number of cells counted.
Explore the reasoning
Pooled: 55/200 = 0.275. Mean per-root index: (0.20 + 0.30)/2 = 0.25. Pooling weights the larger sample more heavily; averaging gives equal weight to each root. State which summary answers your question and retain root-level variation.
Recall this lesson
Lesson recall questions
Question 1 of 5: Mitosis produces two ________ ________ cells used for ________ and asexual reproduction.
Mitosis and the cell cycle
Practice question
Retrieval and explanation · suggested answer
Mitosis produces two ________ ________ cells used for ________ and asexual reproduction.
Try this from memory first.
Beyond the classroomFrom a static slide to cell-cycle researchResearch · cell biology · Enrichment, not required recall
The cell-cycle discoveries recognised by the 2001 Nobel Prize connect simple observations of dividing cells with questions about regulation. A static slide provides useful evidence but cannot by itself measure the speed of each living cell.
Back to the spec: 3.14–3.16: relate observed stage frequencies to mechanisms and experimental limitations.
Nobel Prize: the 2001 Physiology or Medicine awardQuestion
What would a time-lapse method add to a root-tip snapshot?
Explore a possible response
It could follow individual cells and measure transitions and completed divisions directly, provided the imaging conditions do not disrupt their behaviour.