mitotic index experiment

Practical Biology

A collection of experiments that demonstrate biological concepts and processes.

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Investigating mitosis in allium root tip squash, class practical.

Talking about what chromosomes do during mitosis could be very interesting, but seeing them for yourself adds an extra dimension. There are several protocols available for this work and it is notoriously unreliable – often you will not find many dividing cells at all.

This protocol has been tested by the Practical Biology website development team and brings together ideas from SAPS, contributions to the Biotutor discussion list from current teachers, and material from Nuffield Revised Advanced Biology (Longman, 1986).

Lesson organisation

The allium roots need to be prepared 1-10 days in advance of the lesson. Some practitioners report that cutting the root tips around noon makes a difference to the mitotic index, so you may want your technician to cut and ‘fix’ the tips in ethanoic alcohol rather than ask your students to carry out this step.

If you have access to a video microscope it is worth capturing some images, as this procedure can be frustrating.

Apparatus and Chemicals

For each group of students:.

Water bath at 60 °C

Hydrochloric acid, 1 M, 10-25 cm 3 per working group ( Note 4 )

Watch glass (or small dish)

Beaker, 100 cm 3 , 2

Mounted needle

Microscope slide

Paper towels

For the class – set up by technician/ teacher:

Alliums with sprouting roots – garlic is often recommended, or onions ( Note 1 )

Ethanoic alcohol (in dropper bottles) – 3:1 absolute ethanol: glacial ethanoic acid ( Note 2 )

Stain, in dropper bottles, 1 per group ( Note 3 )

Health & Safety and Technical notes

Take care with ethanoic alcohol, hot hydrochloric acid, and with the stain. Wear eye protection when handling these. Carry scalpels with care – hold them on a tile as you walk around the laboratory.

Read our standard health & safety guidance

1 Garlic cloves are most often suggested as a source of root tips. Fresh garlic will sprout overnight if a clove is supported so that it just touches the surface of some water in a vessel – either by simply inserting the clove firmly into a test tube (or boiling tube) of suitable diameter, or by sticking a cocktail stick through the middle of the clove and supporting on the neck of a wider vessel, or cutting holes in expanded polystyrene sheet and inserting the cloves before floating the polystyrene on the surface.

Set up some roots to sprout 10 days before use, some 5 days before use, some 2 and some 1 day before use so that students can compare the rate of mitosis in young and older root tips.

Some practitioners report that cutting the root tips at close to midday makes a difference to the number of dividing cells, so you may want your technician to cut and ‘fix’ the tips in ethanoic alcohol before use. Roots that have been set to sprout for 2 to 5 days seem to give the highest likelihood of finding actively dividing cells.

Alternatives to garlic include onions, hyacinths or seedlings of beans or peas – as long as they are healthy and fast growing.

2 Ethanoic alcohol (“Farmer’s fluid”) is 3 parts absolute ethanol (highly flammable) to 1 part glacial ethanoic acid (Hazcard 38 A describes this as flammable and corrosive). Mix just before use, adding the acid to the alcohol. You can use ethanol IDA or 95% ethanol instead (Hazcard 40 A – highly flammable and harmful due to the presence of methanol), but chromosomes may not be as clearly defined. Root tips can be kept in this fluid for several months.

Ethano-orcein stain: Grind 1.5 g of solid orcein (described on Hazcard 32 as low hazard) with a pestle and mortar. In a fume cupboard, mix 90 cm 3 of glacial ethanoic acid (Hazcard 38A describes this as flammable and corrosive) with 110 cm 3 of distilled water and bring to the boil. Pour the boiling mixture over the orcein and stir very thoroughly (still in the fume cupboard). Leave overnight, then filter and store in a tightly-stoppered dark bottle. If it overstains, dilute with 45% ethanoic acid and try again.

Propionic orcein (made with propionic acid rather than ethanoic) has the advantage that it evaporates more slowly, and is sometimes suggested for this practical.

Toluidine blue – Hazcard 32 describes this as low hazard. Dissolve 0.5 g of solid toluidine blue in 100 cm 3 of water.

4 Hydrochloric acid: Hazcard 47A recommends eye protection when working with hydrochloric acid at 1.0 M. If the acid is hot (60 °C) this is especially important.

5 Whichever stain or squashing technique you use, avoid excess stain or pieces of tissue will drift to the edge of the coverslip and be lost. If you introduce too many air bubbles, add more stain after squashing, using a fine dropping pipette.

6 You can delay squashing for several hours. This allows the cells to take up the stain and to harden, which reduces the chance of them bursting.

SAFETY: Ethanoic ethanol is corrosive, so wear eye protection (goggles). Ethano-orcein stain contains ethanoic acid and also requires you to wear goggles when it is dispensed. You should wear eye protection when handling hot hydrochloric acid. Take care with scalpels and always carry them on a white tile.

Preparation

a Cut off 1-2 cm of the root tips. Put in a small volume of ethanoic acid on a watchglass (or other shallow dish) for 10 minutes. b Meanwhile, heat 10-25 cm 3 of 1 M hydrochloric acid to 60 °C in a water bath.

c Wash the root tips in cold water for 4-5 minutes and dry on filter paper.

d Use a mounted needle to transfer the root tips to the hot hydrochloric acid (see b ) and leave for 5 minutes.

e Wash the root tips again in cold water for 4-5 minutes and dry on filter paper.

f Use the mounted needle to remove two root tips onto a clean microscope slide.

g Cut each about 2 mm from the growing root tip. Discard the rest, but keep the tips ( Note 2 ).

h Add a small drop of stain and leave for 2 minutes ( Note 3 and Note 5 ).

i Break up the tissue with a mounted needle.

j Cover with a coverslip and squash ( Note 6 ) using method A or method B below.

Method A  Place the slide and coverslip on a double layer of paper towel and fold the paper over the coverslip. Make certain that the slide is on a flat surface and squash down on the coverslip with a strong vertical pressure, using your thumb. Do not twist or roll the thumb from side to side Method B  Tap the coverslip about 20 times by dropping a wooden mounted needle or a pencil, blunt end down, from a height of about 5 cm onto the middle of the coverslip.

Investigation

k View the root tips under a microscope (x400 magnification) and look for the chromosomes within cells which are actively dividing.

l Locate the meristematic zone, which has small, apparently ‘square’ cells with nuclei which are large relative to the whole cell area.

The duration of each stage of mitosis has been recorded and the data (see table below) could be used to compare the observed frequencies of the different stages as recorded by students.

These two images show the view that is typical when exploring an allium root tip squash. These slides are of root tips that were cut and fixed after 2 days of growing.

The first is a low power image, and the second a high power close up of a portion of the same slide.

http://www-saps.plantsci.cam.ac.uk This link is to the home page of the Science and Plants for Schools (SAPS) website with links to their teaching resources.  (Website accessed October 2011)

Onion Root Tip Mitosis Stages, Experiment and Results

Mitosis refers to a type of cell division (cell cycle) through which the cell (parent cell) produces two identical daughter cells. Unlike meiosis, which is also a type of cell division , mitosis results in the production of two diploid daughter cells. The two daughter cells contain the same number of chromosomes as the parent cell.

Given that the process results in the proliferation of cells, it's important for general growth and replacement of damaged cells (e.g. the wound healing process).

Growing onion roots

Requirements, sample preparation.

·       Glycerin 

·       Stop clock 

·        Using a clean blade, sharp scissors or scalpel, cut off the tips of the roots (about 5mm in length)

·        Place a cap/lid onto the vial (ensure that the cap/lid has a pinprick hole) and place the vial in the water bath (at 55 degrees C) for about 5 minutes - This enhances the staining process

·        Using a dissecting needle, you can gently mash/squash the root tips to spread out the cells on the glass slide - This prevents several layers of cells from overlapping which would otherwise affect the quality of results

·        Cover the sample (root tip) with a coverslip and gently press the coverslip down, then examine the slide under the microscope starting with low magnification 

Under 10X magnification

*  Generally, a row of cells (single layer) may consist of between 2 and 5 cells.

Higher magnification (stages of mitosis)

The following is a diagrammatic representation of an onion root tip cell during prophase:

Sister chromatids, which contain the same genetic information, are attached at a region known as the centromere which gives the structure an X shape.The kinetochore, which is the site at which microtubules join the chromosomes is also located at the centromere.

*  As the chromatins coil, it becomes increasingly compact which allows the chromosomes to become more visible when viewed under high magnification. 

*  It's also during prophase that spindle microtubules (mitotic spindle) start forming near the nucleus. 

Prometaphase is the second stage of mitosis (it has also been referred to as late prophase). This stage is characterized by increased condensation of chromosomes as well as the breakdown of the nuclear envelope (nuclear membrane). 

*  In late metaphase, the pulling actions of the microtubules, as well as the centrioles, result in the kinetochores (the region at which spindle fibers attach to the chromosomes) facing different directions.

Based on microscopic studies, sister chromatids have been shown to separate and move to the opposite poles of the cell at a rate of between 0.2 and 4 um per minute. Initially, polymerization and depolymerization of microtubules was thought to result in the separation and movement of chromatids to the opposite poles of the cell. 

*  Some of the other factors suggested to contribute to the separation of sister chromatids include the pulling actions of astral microtubules (pulling the poles apart) while interpolar microtubules slide past each other.

Cytokinesis

Cytokinesis refers to the process through which the cytoplasm separates as the cell divides into two identical daughter cells. Unlike animal cells, plant cells have a rigid cell wall that prevents the cell from easily pinching apart to form two identical daughter cells. 

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Mitotic Index Analysis

Series: Methods In Molecular Biology > Book: Chromosome Analysis

Protocol | DOI: 10.1007/978-1-0716-2433-3_3

• Department of Pediatrics and Human Development & Department of Pharmacology & Toxicology, Michigan State University, East Lansing, MI, USA
• Department of Environmental & Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA

Cellular division is a fundamental process of cellular growth. First, cells replicate their DNA in S phase and then undergo mitosis which, under normal conditions, leads to complete cell division. Moreover, mitotic activity correlates to cellular

Cellular division is a fundamental process of cellular growth. First, cells replicate their DNA in S phase and then undergo mitosis which, under normal conditions, leads to complete cell division. Moreover, mitotic activity correlates to cellular growth activity. The simplest and classical method to measure mitotic activity (mitotic index (MI)), is the manual counting of mitotic cells among a given cell population of interest. The latter can be accomplished via phase contrast microscope observation. However, Giemsa staining may improve accuracy and consistency. Fluorescence immunostaining targeting specific phosphorylations of proteins at critical cell cycle steps will provide further improved analysis via high-throughput capacity of flow or imaging cytometer. Finally, time lapse image analysis provides quantitative and qualitative metrics delineating the process of cellular division including timing of division, duration of mitosis, and failure to procced through or complete mitosis.

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Citations (4)

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• Howard A, Pelc S (1953) Synthesis of deoxyribonucleic acid in normal and irradiated cells and its relation to chromosome breakage. Heredity 6(supple):261–273
• Hoffman JG (1947) Wright's hypothesis: its relation to volume growth of tissue cells and mitotic index. Science 106(2754):343–344. https://doi.org/10.1126/science.106.2754.343-a
• Hoffman JG (1949) Theory of the mitotic index and its application to tissue growth measurement. Bull Math Biophys 11(2):139–144. https://doi.org/10.1007/BF02477499
• Kim JY, Jeong HS, Chung T, Kim M, Lee JH, Jung WH, Koo JS (2017) The value of phosphohistone H3 as a proliferation marker for evaluating invasive breast cancers: a comparative study with Ki67. Oncotarget. 8(39):65064–65076. https://doi.org/10.18632/oncotarget.17775
• Medri L, Volpi A, Nanni O, Vecci AM, Mangia A, Schittulli F, Padovani F, Giunchi DC, Zito A, Amadori D, Paradiso A, Silvestrini R (2003) Prognostic relevance of mitotic activity in patients with node-negative breast cancer. Mod Pathol 16(11):1067–1075. https://doi.org/10.1097/01.MP.0000093625.20366.9D
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Staining a Root Tip and Calculating Its Mitotic Index

In this practical activity students stain root tips and examine them for signs of cells dividing by mitosis. Students can either compare two different sources of root tip or two different stains. The mitotic index is the fraction of cells in a microscope field which contain condensed chromosomes. This index can be calculated for each slide prepared

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Mitotic Index Analysis

Affiliations.

• 1 Department of Environmental & Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA. [email protected].
• 2 Department of Pediatrics and Human Development & Department of Pharmacology & Toxicology, Michigan State University, East Lansing, MI, USA.
• PMID: 36066706
• DOI: 10.1007/978-1-0716-2433-3_3

Cellular division is a fundamental process of cellular growth. First, cells replicate their DNA in S phase and then undergo mitosis which, under normal conditions, leads to complete cell division. Moreover, mitotic activity correlates to cellular growth activity. The simplest and classical method to measure mitotic activity (mitotic index (MI)), is the manual counting of mitotic cells among a given cell population of interest. The latter can be accomplished via phase contrast microscope observation. However, Giemsa staining may improve accuracy and consistency. Fluorescence immunostaining targeting specific phosphorylations of proteins at critical cell cycle steps will provide further improved analysis via high-throughput capacity of flow or imaging cytometer. Finally, time lapse image analysis provides quantitative and qualitative metrics delineating the process of cellular division including timing of division, duration of mitosis, and failure to procced through or complete mitosis.

Keywords: Histone H3 serine residue 10; Mitotic index; Time lapse image analysis.

© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

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• Howard A, Pelc S (1953) Synthesis of deoxyribonucleic acid in normal and irradiated cells and its relation to chromosome breakage. Heredity 6(supple):261–273
• Hoffman JG (1947) Wright's hypothesis: its relation to volume growth of tissue cells and mitotic index. Science 106(2754):343–344. https://doi.org/10.1126/science.106.2754.343-a - DOI - PubMed
• Hoffman JG (1949) Theory of the mitotic index and its application to tissue growth measurement. Bull Math Biophys 11(2):139–144. https://doi.org/10.1007/BF02477499 - DOI - PubMed
• Kim JY, Jeong HS, Chung T, Kim M, Lee JH, Jung WH, Koo JS (2017) The value of phosphohistone H3 as a proliferation marker for evaluating invasive breast cancers: a comparative study with Ki67. Oncotarget. 8(39):65064–65076. https://doi.org/10.18632/oncotarget.17775 - DOI - PubMed - PMC
• Medri L, Volpi A, Nanni O, Vecci AM, Mangia A, Schittulli F, Padovani F, Giunchi DC, Zito A, Amadori D, Paradiso A, Silvestrini R (2003) Prognostic relevance of mitotic activity in patients with node-negative breast cancer. Mod Pathol 16(11):1067–1075. https://doi.org/10.1097/01.MP.0000093625.20366.9D - DOI - PubMed
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The Biology Corner

Biology Teaching Resources

Investigation: Mitosis

This mitosis investigation was created during the 2020 pandemic for remote learning. In previous years, biology students would view slides in the lab and analyze data on cancer and mitotic index.

This at-home activity only looks at the mitosis of onion cells and I plan to add cancer and mitotic index as a separate activity. I used a microscope camera to take photos of onion root slides where students can count the number of cells in each phase

Then they use those numbers to calculate the amount of time spent in each. Students currently need to click on a link to a Google folder that contains the five photos though I may eventually move them to something more interactive.

You are welcome to download them and use them in your own class or even add them to the google slides so students do not need to leave that page.

The activity contains introductory information on the types of cells that divide (embryos, meristems) and the phases of mitosis as a review. Students are shown how to identify the phases on the photographs of the root tips. Then they complete a table which counts the number of cells in interphase, prophase, metaphase, anaphase, and telophase.

A simple formula uses these numbers to calculate the time spent based on the known time it takes for these cells to complete the cell cycle (720 minutes).

For a more advanced activity that uses real slides, check out the lab on Mitosis and Cancer.

Shannan Muskopf

• Biology Article

Study Of Mitosis In Onion Root Tip Cells

Aim of the experiment.

To study and demonstrate mitosis by preparing the mount of an onion root tip cells.

Theory Of The Experiment

For entities to mature, grow, maintain tissues, repair and synthesize new cells, cell division is required. Cell division is of two types:

In mitosis , the nucleus of the Eukaryotic cells divides into two, subsequently resulting in the splitting of the parent cells into two daughter cells. Hence, every cell division involves two chief stages:

• Cytokinesis – Cytoplasm division
• Karyokinesis – Nucleus division

Stages Of Mitosis

The various stages of mitosis are:

1. Prophase

• The process of mitosis is initiated at this stage wherein coiling and thickening of the chromosomes occurs
• Shrinking and hence the disappearance of the nucleolus and nuclear membrane takes place
• The stage reaches its final state when a cluster of fibres organizes to form the spindle fibres

2. Metaphase

• Chromosomes turn thick in this phase. The two chromatids from each of the chromosomes appear distinct
• Each of the chromosomes is fastened to the spindle fibres located on its controller
• Chromosomes align at the centreline of the cell

3. Anaphase

• Each of the chromatid pair detaches from the centromere and approaches the other end of the cell through the spindle fibre
• At this stage, compressing of the cell membrane at the centre takes place

4. Telophase

• Chromatids have reached the other end of the cell
• The disappearance of the spindles
• Chromatin fibres are formed as a result of uncoiling of daughter chromosomes
• The appearance of two daughter nuclei at the opposing ends due to the reformation of the nucleolus and nuclear membrane
• At this phase, splitting of the cell or cytokinesis may also occur

Post mitosis, the next stage is referred to as interphase, which is part of the cell cycle that is non-dividing and between two consecutive cell divisions . A cell spends most of its life in the interphase. It comprises the G1, S and G2 stages.

Why is onion root tip used to demonstrate mitosis in this experiment?

It is because of the meristematic cells that are situated in the tip of the roots that render the most desirable and suitable raw material to study the different stages of mitosis. Onion is a monocot plant. Monocotyledonous plants possess large chromosomes that are clearly visible. Hence, their root tips are used. The period of time taken for mitosis varies as it is dependent on the cell type and type of species.

Is mitosis influenced by any factor? If yes, name them.

Yes, mitosis, the cell cycle is affected by various factors such as time and temperature.

Materials Required

• Compound microscope
• Acetocarmine stain
• N/10 Hydrochloric acid
• Filter paper
• Aceto alcohol (Glacial acetic acid and Ethanol in the ratio 1:3)
• Glass Slide
• Onion root peel
• Watch glass

Procedure Of The Experiment

• Place an onion on a tile
• With the help of a sharp blade, carefully snip the dry roots of the onion
• Place the bulbs in a beaker containing water to grow the root tips
• It may take around 4 to 6 days for the new roots to grow and appear
• Trim around 3 cm of the newly grown roots and place them in a watch glass
• With the help of forceps, shift it to a vial holding freshly prepared aceto-alcohol i.e., a mixture of glacial acetic acid and ethanol in the ratio 1:3
• Allow the root tips to remain in the vial for one complete day
• With the help of forceps, pick one root and set in on a new glass slide
• With the help of a dropper, allow one drop of N/10 HCl to come in contact with the tip of the root. Additionally, add around 2 to 3 drops of the acetocarmine stain
• Heat it lightly on the burner in such a way that the stain does not dry up
• Excessive stain can be carefully treated using filter paper
• The more stained part of the root tip can be trimmed with the help of a blade.
• Discard the lesser stained part while retaining the more stained section
• Add a droplet of water to it
• With the help of a needle, a coverslip can be mounted on it
• Gently tap the coverslip with an unsharpened end of a needle in order for the meristematic tissue of the root tip present under the coverslip to be squashed properly and to be straightened out as a fine cell layer
• The onion root tip cells’ slide is now prepared and ready to be examined for different stages of mitosis
• Observe and study mitosis by placing the slide under the compound microscope. Focus as desired to obtain a distinct and clear image

Observations and Conclusion

• The slide containing the stained root tip cells is placed on the stage of the compound microscope, changes taking place are noted and sketched.
• The different phases of mitosis, such as prophase, metaphase, anaphase and telophase can be observed.

Viva Questions

Q.1. Why is mitosis also referred to as the equational division?

A.1. It is because the chromosome  number present in the daughter cells is the same as the number of chromosomes present in the parent cell.

Q.2. To study mitosis, what is the best time to harvest onion root tips and why?

A.2. Early morning is the best time as the root tips actively undergo cell division in the morning. When such a material is used, all stages of cell division can be observed and studied.

Q.3. Other than an onion, can you suggest any other raw material for the study of mitosis.

A.3. Dividing cells of these can be picked:

• Shoot apex of plants
• Cells from the root tips of any herbaceous plants
• Gills of fish (epithelial cells)
• Tadpole larvae (tail)

Q.4. For the cytological study, why are different parts of monocots preferred?

A.4. Since they possess larger chromosomes that are clearly visible under a light microscope.

Q.5. Why is the stain acetocarmine used in this experiment?

A.5. This stain is used to study chromosomes as it stains them in a deep red tint without staining the cytoplasm.

Q.6. Where does the spindle fibre originate from?

A.6. The spindle fibres originate from:

• In-plant cells – Cytoplasm
• In animal cells – Centrioles

Q.7. Mention the phase of the cell division in which chromosomes are observed distinctly.

A.7. Metaphase. They are shortest and thickest in this stage and in their condensed form.

Q.8. During metaphase, which chemical can be used to stop the cell division process?

A.8. The spindle fibre formation can be inhibited by Colchicine during this stage.

Q.9. Where is colchicine obtained from?

A.9. Colchicum autumnale plant belonging to the Liliaceae family.

Q.10. Where does mitosis occur?

A.10. This type of cell division takes place in the vegetative cells.

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• Cell Prolif
• v.38(2); 2005 Apr

Quantification of cell‐cycle distribution and mitotic index in Hydra by flow cytometry

1 Department of Biochemistry, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil and

A. Tárnok

2 Pediatric Cardiology, Cardiac Center Leipzig, University of Leipzig, Germany

Abstract.  The applicability of flow cytometry (FCM) to analyse cell‐cycle distribution and mitotic cells in Hydra oligactis and Hydra vulgaris is demonstrated. The freshwater polyps H. vulgaris and H. oligactis are well‐accepted animal models for studying cell proliferation, regeneration and differentiation. Disintegrated animals were labelled for FCM analysis according to the method of Nuesse et al . [(1990) Flow cytometric analysis of G 1 and G 2 /M‐phase subpopulations in mammalian cell nuclei using side scatter and DNA content measurements. Cytometry 11 , 813]. Proliferation and regeneration experiments, in the absence or presence of the oligopeptide head activator, were quantified. Cell‐cycle analysis of different parts of the animals shows low proliferation in the head region and high proliferation in the gastric and foot regions. Cell‐cycle analysis of different parts of Hydra , comparison of H. oligactis and H. vulgaris , as well as pharmacological treatment, yielded results that are in agreement with prior microscopic analysis. Our results demonstrate that FCM is an appropriate technique for quantifying proliferation in this animal model. It can be used for basic research on development, regeneration and differentiation as well as for innovative drug investigation and toxicology studies.

INTRODUCTION

Freshwater polyps of Hydra ( Hydra vulgaris , former Hydra attenuata , and Hydra oligactis , former Hydra fusca ) belong to the metazoan phylum Cnidaria, are believed to be one of the most ancient animal groups and are the first in evolution of developed specialized tissues. The fresh‐water polyp Hydra is a well‐accepted animal model for the study of cell proliferation and differentiation (reviewed by Meinhardt 1996 ; Galliot & Schmid 2002 ; Holstein et al . 2003 ). Hydra has a bipolar cellular organization along a single axis of symmetry. Because of its high capacity for regeneration, Hydra is a suitable system for studying morphogenetic processes, not only in developing but also in regenerating animals ( Müller 1996 ). Polarity is generated by two types of gradient and is maintained even during regeneration of new animals from very small pieces ( Bode & Bode 1984 ).

Besides its major impact on developmental biology research for many years, Hydra has become of striking importance as a model organism. The successful application of molecular techniques has linked insights into molecular circuitry with cell proliferation and differentiation events. These breakthroughs include the discovery of transcription factor genes and the generation of knock‐out phenotypes using antisense and RNA interference techniques ( Fenger et al . 1994 ; Cardenas & Salgado 2003 ; Kaloulis et al . 2004 ).

Hydra can be compared in many regards with an early‐stage embryo, as its body consists of three stem cell populations that are retained through the lifetime of the animal: ectodermal, endodermal and interstitial cells. Hydra species have therefore become important test organisms for environmental pollutants and drug treatment response ( Arkhipyhuk & Malinovskaya 2002 ; Karntanut & Pascoe 2002 ; Pascoe et al . 2002 ; Lum et al . 2003 ).

Epithelial stem cells can either differentiate into head‐ or foot‐specific cells. Interstitial cells consist of pluripotent stem cells and precursor cells which can differentiate into neurones, nematocytes and gland cells. They are uniformly distributed along the body column but are absent from head and foot tissue. The formation of head‐specific tissue is regulated by a head activator (HA), which has been isolated from Hydra tissue ( Bodenmüller & Schaller 1981 ). Besides differentiating epithelial cells into head‐specific tissues, HA also acts as a mitogen, increasing the number of S‐phase cells about 12 h after stimulation ( Schaller 1976 ). Most organisms regulate their cell cycle by check points in the G 0 /G 1 ‐phase. In Hydra , however, terminal differentiation of either head‐ or foot‐specific ectodermal epithelial cells starts and terminates in the G 2 ‐phase, implicating restriction points for cell‐cycle regulation in the G 2 ‐phase ( Dübel & Schaller 1990 ). Proliferation events have been mainly accessed by microscopic counting of mitotic nuclei following Hoechst 33258 staining, by determining the nuclear content of single cells using a microscope photometer, or by pulse labelling with 5′‐bromo‐2′‐deoxyuridine (BrdU) and staining with an anti‐BrdU antibody ( Dübel et al . 1987 ; Dübel 1989 ). In all of these cases, for statistical relevance large populations of cells needed to be analysed, making the methods time consuming.

In the present study, we demonstrate the applicability of flow cytometry (FCM) to measure cell cycle and proliferation in individual Hydra cells that were obtained by disintegration of whole Hydra individuals or of their different body‐parts. The technique can be used conveniently to analyse proliferation events in this important animal system during HA‐induced cell proliferation or regeneration events after dissecting live animals. We show here that the results of the cell‐cycle distribution are in accordance with previous microscopic evaluation (David & Campbell 1972; Gierer et al . 1972 ; Campbell & David 1974 ; David & Gierer 1974 ; David & Murphy 1977 ; Dübel et al . 1987 ; Dübel & Little 1988 ; Hoffmeister 1991 ). The technique is time saving and an appropriate tool for quantification of small differences between stimulated and unstimulated cells as the cell‐cycle distribution of large populations can be analysed.

MATERIALS AND METHODS

Hydra culture.

Hydra vulgaris and Hydra oligactis were cultured in 0.5 m m sodium phosphate, 1 m m CaCl 2 , 0.1 m m MgCl 2 , 0.1 m m KCl, pH 7.6 (this and all following reagents from Sigma‐Aldrich, Deisenhofen, Germany). They were fed daily with nauplii of Artemia salina and washed 6 h after feeding. Prior to experiments Hydras were starved for 1 day.

Disintegration of whole animals or parts of animals

For determination of cell‐cycle distributions, either whole animals ( Fig. 1 : schematic morphology of Hydra ) were used or animals dissected in head, stomach and foot, followed by mechanical disintegration into single cell suspensions ( Gierer et al . 1972 ). To this end, animals were transferred into isolation medium (KCl 3.6 m m , CaCl 2  6 m m , MgSO 4  1.3 m m , Na‐citrate 6 m m , Na‐pyruvate 6 m m , glucose 6 m m , TES buffer, 12.5 m m , phenol‐red 100 mg/l, in distilled water at pH 6.9, 70 mOsm). For disintegration, 100 animals or 100 parts of animals were then placed in a plastic tube with 500 µl of isolation medium containing 7% acetic acid, 7% glycerol and 2.5 mg/ml pronase and pipetted through glass pipettes with subsequently decreasing gauge sizes starting at about 1‐mm diameter and decreasing to about 100 µm. Prior to use, pipette tips were melted to decrease the opening size and to remove sharp edges.

Morphology of Hydra . Microscope image of Hydra vulgaris : 1. tentacle; 2. hypostome; 3. nematocytes; 4. gastrum; 5. foot. Dotted lines indicate where the animals were cut for head, gastric and foot samples. Scale bar indicates 100 µm.

Determination of cell‐cycle distribution and mitotic index by FCM

The single cell suspensions obtained were used for cell nucleus preparation, DNA staining with PI and subsequent cell‐cycle analysis by FCM according to the method of Nuesse et al . (1990 ). In brief, isolated cells were spun down (400  g , 5 min) and roughly 10 6  cells were suspended in 500 µl staining solution I (NaCl 10 m m , NA‐citrate 1.7 m m , RNase 10 µg/ml, Nonidet P‐40 0.03%, PI 60 µ m ). After 30 min incubation at room temperature, staining solution II (citric acid 1.5%, sucrose 0.25  m , PI 90 µ m ) was added, and nuclei were stained with PI, on ice for 1 h. Before analyses by FCM, the suspension was filtered through a nylon filter, mesh size of 50 µm, to remove clumps.

FCM analysis was performed on an EPICS 751 cell sorter (Beckman‐Coulter, Hialeah, FL, USA) that was equipped with an argon‐ion laser tuned to the 488 nm emission line (laser power 50 mW; Coherent, Palo Alto, CA, USA). PI was excited and the emitted PI fluorescence was collected by a 590‐nm long‐pass filter (Omega Optical, Brattleboro, VT, USA). Side‐scatter signals were measured by means of a dichroic 500 nm long‐pass mirror, reflected blue scattered light, and detected by a 488‐nm band‐pass filter. Per sample, data of 20 000 nuclei were collected. Data were analysed offline using the DAS software, kindly provided by Dr W. Beisker ( Beisker 1994 ).

All experiments were carried out at a constant temperature of 18 °C. For regeneration assays, gastric regions of animals were cut and then incubated for 3 h in culture medium alone or, in order to block cells in mitosis, in culture medium containing 0.5 µg/ml nocodazol ( Dübel & Little 1988 ). Proliferation experiments with the Hydra neuropeptide HA ( Schaller 1976 ) were performed as follows: synthetic HA (pGlu‐Pro‐Pro‐Gly‐Gly‐Ser‐Lys‐Val‐Ile‐Leu‐Phe, Bachem, Bubendorf, Switzerland) was solubilized in trifluoroethanol and lyophilized in 5‐nmol samples. Lyophilized HA samples were re‐suspended in 250 µl 3M ammonium sulphate and 250 µl distilled water and then diluted to its final concentration in Hydra culture medium. For determination of cell‐cycle distribution and percentage of cells undergoing mitosis, sliced gastric regions were incubated for 3 h in the absence or presence of HA (10 −12 to 10 −9   m ). Then gastric regions were disintegrated and cells were stained with PI, used for determination of cell‐cycle distribution, and the percentage of cells in mitosis was determined by FCM. Data analysis was performed as described in Nuesse et al . (1990 ).

Determination of cell‐cycle distribution by FCM in disintegrated cells from Hydra oligactis

Using the preparation and staining method of Nuesse et al . (1990 ), cell‐cycle distributions could be easily analysed by FCM. Cell‐cycle distributions were either determined in disintegrated cells from whole animals or from dissected parts containing head, foot or gastric regions (see Fig. 1 for cut positions). The resulting coefficients of variation for the G 0 /G 1 ‐peak of cell‐cycle distribution were generally below 5% in untreated animals. The fraction of disrupted nuclei appearing as a sub‐G 1 population with low PI fluorescence was dependent on the number of animals that had been used in the experiment for disintegration. This fraction was above 70% of all events if 10 or fewer animals were used. Best results, that is, lowest fraction of nuclear debris, were obtained when at least 100 animals or parts of animals were used for disintegration ( Fig. 2 ).

Quality of the Hydra cell nuclei preparation depends on the number of animals used for disintegration. Increasing numbers of whole Hydra oligactis animals (a, 5; b, 10; c, 50; d, 100) were mechanically disintegrated into single cell suspensions, cell nuclei were prepared as detailed in MATERIALS and METHODS. Cell nuclei were stained with PI for cell‐cycle analysis and determination of the percentage of cell debris. Cell suspensions were measured by FCM. Particles with fluorescence intensity below the dotted lines were considered as cell debris. The fractions of cell debris and intact stained nuclei are given as inserts.

Whole animals or parts of animals displayed differences in their cell‐cycle distributions. Figure 3 shows a representative experiment of Hydra oligactis cell‐cycle distribution. Cells isolated from the head region showed the highest fraction of G 0 /G 1 ‐phase, whereas those from the foot region showed the highest fraction in S‐ and G 2 ‐phase ( Fig. 3 ). These differences are as a result of a high number of nematocytes (mostly G 1 ‐phase) in the tentacles of the head region, of many interstitial cells (i‐cells, stem cells, 50% in S‐phase) in the gastric region and a high number of gland and epithelial cells in the foot (> 50% blocked in G 2 ). The results of the cell‐cycle distribution were in accordance with microscopic evaluations in H. oligactis from the literature as shown in Table 1 . In this table, data published in several studies are summarized ( Gierer et al . 1972 ; Campbell & David 1974 ; Dübel et al . 1987 ; Dübel 1989 ; Zacharias et al . 2004 ). Similarly, FCM data on H. vulgaris were found to be in agreement with already published data based on microscopic counting ( Table 2 ; David & Gierer 1974 ; Dübel & Little 1988 ; Hoffmeister 1991 ).

Cell‐cycle distributions in disintegrated cells from Hydra oligactis (entire animals or dissected parts). Cell suspension obtained from whole animals or parts following cutting of animals in heads, gastric regions and feet were macerated according to Gierer et al . (1972 ), and the single cell suspensions were stained with PI for cell‐cycle measurements. (a–d) Representative measurements of cell‐cycle distributions of whole animals: (a) head regions containing nematocytes, epithelial cells, and nerve cells; (b) gastric regions containing epithelial and interstitial cells; (c) feet regions containing gland and mucous cells, epithelial cells, and nerve cells (d). Cell‐cycle distributions are given as percentage of the whole analysed population. The data are representative for at least three independent experiments. Ranges of measured minimal and maximal values are given in Table 1 .

Comparison of cell‐cycle distribution and mitotic index in H. oligactis determined by flow cytometry (FCM) or microscopy (fluorescence microscopy or microscope photometry, data from various sources, see Results ). Cell‐cycle distributions and mitotic index are given as range of measured minimal and maximal values. (FCM, range of at least three independent experiments). Microscope data from David & Gierer (1974 ), David & Murphy (1977 ), Dübel & Little (1988 ) and Hoffmeister (1991 )

Determination of cell‐cycle distribution and mitotic index in H. vulgaris by FCM or microscopy (data from various sources, see Results ). Cell‐cycle distributions are given as range of measured minimal and maximal values; for mitotic index no comparable data were available from the literature. (FCM, range from at least three independent experiments). Microscopic data from Gierer et al . (1972 ), David & Campbell (1972), Campbell & David (1974 ) and Dübel et al . (1987 )

Increase in G 2 phase and determination of the percentage of cells in mitosis following metaphase block

In order to prove the validity of FCM to determine cell‐cycle distribution and mitotic index, entire H. oligactis specimens were incubated in the absence or presence of nocodazol for 6 h and then disintegrated and analysed for cell‐cycle distribution ( Fig. 4 ). Nocodazol arrests proliferating cells in the G 2 ‐phase ( Dübel & Little 1988 ), resulting in an increase of G 2 /M‐phase cells from 20.1 ± 3.6% (untreated control) to 37.5 ± 3.3% in the presence of 0.5 µg/ml nocodazol ( P  < 0.005, student's t ‐test).

Proliferation block of Hydra oligactis in G 2 /M phase by nocodazol. Gastric regions from Hydra were obtained by dissection. Cuts were carried out as indicated in Figure 1 . Regeneration assays were performed for 6 h in absence or presence of 0.5 µg/ml nocodazol that blocks cell division in the G 2 /M‐phase. Following incubation, the tissues were macerated and cell nuclei were prepared as detailed in MATERIALS and METHODS and were stained with PI. The percentage of cells in G 2 ‐phase was increased because of the mitotic block by nocodazol. Cell‐cycle distributions are given as percentages of all nuclei. In the presence of nocodazol, an increase of cells in G 2 /M phase from 20.1 ± 3.6 to 37.5 ± 3.3% (mean value ± standard error) was observed. (Mean of six independent experiments; P  < 0.05, unpaired student's t ‐test.)

FCM can also be used to determine the mitotic index during regeneration of animals ( Fig. 5 ). In these experiments, the head region of H. oligactis was removed by cutting, and the regeneration of the head by proliferation of cells in the gastric region was measured as an increase of cells in found in mitosis. In order to determine mitotic indices by FCM, a bivariate analysis (DNA versus side‐scatter) was employed according to Nuesse et al. (1990 ), which allowed the separation of mitotic nuclei from other nuclei based on their increased side‐scatter values. The localization of cells undergoing mitosis was verified in experiments where cells were arrested in mitosis by a metaphase block in the presence of nocodazol ( Fig. 5 ; cell population in boxed area). This population was separated by cell sorting and confirmed by microscopic evaluation to contain exclusively mitotic cell nuclei only (data not shown).

Mitotic index in regenerating gastric regions of Hydra oligactis. Bivariate analysis of DNA content versus side scatter ( Nuesse et al . 1990 ) was employed in analogy to mammalian cells, in order to detect mitotic cell nuclei with high 90° side‐scatter signals. Gastric regions were obtained by dissection of entire animals. Regeneration assays were carried out for 2 h with or without 0.5 µg/ml nocodazol. Cells appearing in the boxed area were separated by cell sorting, and sorted cells were identified as mitotic cells by microscopic evaluation (not shown). The data are representative of three independent experiments. Mean values ± standard errors for mitotic indices in the absence and presence of nocodazol were 1.5 ± 0.2 and 5.9 ± 0.4%, respectively (mean of at least three experiments, P  < 0.05).

Detection and quantification of head‐activator (HA)‐induced proliferation in Hydra HA stimulates cell proliferation in Hydra

This was brought about by incubation of freshly cut gastric regions with HA (10 −10   m ) for 3 h. Following incubation, animals were disintegrated and cell nuclei were stained. Figure 6(a and b) show a typical experiment in which cell‐cycle distribution and mitotic indices of cells from untreated gastric regions ( Fig. 6a ) were compared with those which had been treated with HA (10 −10   m ) ( Fig. 6b ) or nocodazol ( Fig. 6c ).

Quantification of head‐activator‐induced proliferation of the gastrum of Hydra oligactis . For induction of cell proliferation in Hydra , gastric regions were kept untreated (a)or were stimulated for 3 h with 10 −10   m head activator (HA) (b) or treated with nocodazol (0.5 µg/ml) for 3 h (c) to arrest cells in mitosis and to localize the mitotic cell population (boxed areas in right panels). The left panels show cell‐cycle distributions of Hydra cells in the presence and absence of nocodazol or HA. In the right panels, mitotic indices of the respective populations were quantified. The mitotic index was determined as the fraction of nuclei in the boxed area over total nuclei.

An increase of the mitotic cell population was found following treatment by HA ( Fig. 6a and b , right panels). The positions of the gating region for mitotic nuclei were again verified in the presence of nocodozal ( Fig. 6c , right panel) followed by cell sorting and microscopic identification of the respective population as cells being in mitosis only (data not shown). Figure 6(a and b) (right panels) shows a representative experiment in which the mitotic index in the presence of HA (10 −10   m ) increased from 2.9 to 3.5%. The average mitotic index in the presence of HA (10 −10   m ) as the result of three independent experiments was 3.8 ± 0.5% compared with the average mitotic index of 2.8 ± 0.4% (mean ± standard deviation) of the untreated controls ( P  < 0.03 as determined by the unpaired student's t ‐test). Using comparable conditions, Schaller (1976 ) used microscopic analysis to determine the increase in mitotic index of interstitial cells following treatment of Hydra specimens with HA. Mitotic indices increased from 2.5 ± 0.5 to 4.1 ± 0.8%, from 2.0 ± 0.5% to 3.4 ± 0.6%, from 1.8 ± 0.7 to 3.5 ± 0.6%, and from 1.5 ± 0.5 to 3.0 ± 0.7% (HA versus untreated control, respectively). The HA‐induced effect on stimulation of mitosis was dose dependent, reaching maximal values at HA concentrations of 10 −11 , decreasing at 10 −9 and 10 −10   m HA ( Fig. 7 ). Increases in mitosis in the presence of 10 −11 and 10 −10   m HA versus control were significant (student's t ‐test P  < 0.05).

Determination of dose dependence of HA‐induced stimulation of H. oligactis mitotic index by flow cytometry. Proliferation stimulation assays of Hydra single‐cell suspensions were performed for 2 h in the absence (control) or presence of HA (10 −10 to 10 −9   m ). Percentages of cells in mitosis were determined as detailed in legends to ​ to5, 5 , ​ ,6 6 and in MATERIALS and METHODS. Data are mean values ± standard error of at least three independent experiments. The increases in mitotic index obtained in the presence of 10 −11 and 10 −10   m HA versus control were statistically significant ( P <  0.05).

The obtained results are in accordance with the results of Neubauer et al . (1990) , who characterized a high‐affinity HA receptor inducing proliferation at very low concentration of HA (> 10 −13   m ) and differentiation at higher HA concentrations (> 10 −11   m ). It is worth mentioning that the increase in percentage of mitosis, induced by HA ( Figs 6a and b , right panels), correlates with a minor decrease in the percentage of cells in G 2 ‐phase and an increase of those in G 1 ‐phase ( Figs 6a and b , left panels), as proliferating cells already accumulated in G 2 ‐phase pass through mitosis ( Dübel et al . 1987 ; Dübel 1989 ). We have shown that analysis of a relatively small increase of mitotic index in Hydra , as caused by HA ( Fig. 6a and b , right panels) can be quantified by analysing a large number of cells by FCM.

Comparison of cell‐cycle distribution of different parts of Hydra oligactis and Hydra vulgaris

We have determined cell‐cycle distribution for entire animals and body parts of H. oligactis and H. vulgaris ( ​ (1, 1 , ​ ,2). 2 ). In the head region of both species, most cells are in the G 1 ‐phase (up to 70% of the whole population), whereas less than 30% are in G 2 ‐phase. Proliferation is increased in the gastric and foot regions, as determined by the percentage of cells in S‐phase. Maximal rates of proliferation and G 2 were found in the foot region where up to 60% of the entire cell population were in S‐ and G 2 ‐phase. In accordance with cell‐cycle distributions, the mitotic index was considerably higher in the gastric and the foot region of H. oligactis when compared with the head region. The discrepancy with microscopic data may result from the enormous number of cells which were analysed by FCM compared with the relatively low number of cells counted by microscopic analysis. The data for cell‐cycle distribution largely agree with findings based on microscopic evaluations listed in ​ in1, 1 , ​ ,2. 2 . As an example, H. oligactis as a whole contain less S‐phase cells than H. vulgaris (mean FCM: 23 versus 16%; microscope: 29 versus 20%). Data for H. oligactis cell‐cycle and mitotic index are from the following sources ( David & Gierer 1974 ; David & Murphy 1977 ; Dübel & Little 1988 ; Hoffmeister 1991 ). Cell cycle distribution in H. vulgaris has been investigated by the following workers (David & Campbell 1972; Gierer et al . 1972 ; Campbell & David 1974 ; Dübel et al . 1987 ).

We have developed a flow cytometric method, capable of rapidly determining cell proliferation events of disintegrated cells from intact H. oligactis and H. vulgaris , or their body parts, and from animals exposed to external stimuli. Hydra have a simple body plan with a head and tentacles at one side and a foot at the opposite end of a gastric column (see Fig. 1 ). In the intact animal, proliferation occurs as epithelial layers are continuously displaced along the body column. We have determined cell‐cycle distribution and mitotic indices of Hydra and found them to be in accordance with data obtained by BrdU labelling or mitotic cell counting. In contrast to microscopic evaluations of proliferation, FCM has the advantage that large populations of cells can be monitored in a very short time period, allowing the determination of small changes with high accuracy.

FCM has been extensively used to study cell‐cycle distribution and proliferation in mammalian cells. Furthermore, in invertebrates, cells can disintegrate from complex organisms, for example, male accessory glands of mealworm pupae ( Happ et al . 1985 ) and protozoa such as African trypanosomes and malaria parasites ( Jacobberger et al . 1992 ; Mutomba et al . 1997 ). These now can be analysed conveniently to determine their cell‐cycle distribution. Protocols for staining cell nuclei and analysis by flow cytometry have been extended to use for plant cells ( Sans et al . 1997 ; Lucretti et al . 1999 ). However, to the best of our knowledge, cell‐cycle analysis by FCM has not been used previously for Hydra species.

We have shown here that cell‐cycle distribution of Hydra cells can be analysed easily by FCM. The fraction of cell nuclei in mitosis can be determined by using the method of Nuesse et al . (1990 ), which was originally developed to measure mitosis in mammalian cells. This method is based on the detection of high scattering properties of cells in G 2 phase undergoing mitosis. The percentage of this fraction over the total cell population was increased when the cells had been treated with the metaphase blocker nocodazol during cell proliferation. The mitotic population with high light scattering characteristics could be clearly separated from the remaining population by cell sorting. Our FCM data on cell cycle and proliferation of Hydra oligactis and Hydra vulgaris , and microscopic data from others sources (David & Campbell 1972; Gierer et al . 1972 ; Campbell 1974 ; David & Gierer 1974 ; David & Murphy 1977 ; Dübel et al . 1987 ; Dübel & Little 1988 ; Hoffmeister 1991 ) (see ​ (see1, 1 , ​ ,2) 2 ) were largely in agreement. Discrepancy of data collected by FCM from microscopic data, such as mitotic indices and percentages of cells in G 2 ‐phase isolated from entire animals, can be explained by the different numbers of analysed cells. The huge number of cells analysed by FCM also makes small differences between two data sets statistically relevant. For instance, HA‐induced proliferation of Hydra cells yields an increase of mitotic index up to 50% above baseline.

In summary, FCM is an appropriate technique for quantification of cell proliferation by measuring cell‐cycle and mitotic index at the same time. This technique has already been shown to be extremely useful for detection of cell proliferation in neoplasms as well as in vitro cultures. Hydra is not only a model for early embryogenesis, but also demonstrates remarkable regenerative capabilities which appear to be conserved over large phylogenetic stretches with evidence for a homologue origin of regenerative capability (reviewed by Löwenheim 2003 ). In view of that, our assay, rapid analysis of proliferation by FCM, following modulation of gene expression could be helpful for linking activation of certain genes with biological function. Furthermore, this new approach should enable rapid high‐throughput analysis of the effects of environmental pollutants and drugs on regeneration capability of using Hydra as a biosensors.

ACKNOWLEDGEMENTS

The authors wish to thank Prof. H. C. Schaller in whose laboratory this work was carried out and Dr S. A. Hoffmeister‐Ullerich for providing us with Hydra vulgaris and Hydra oligactis . HU is currently supported by a grant from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and a fellowship from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), Brazil.

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Mitotic Index

• First Online: 18 August 2018

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• Elisa Graña 3  

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The cell division cycle is a highly controlled process, essential for plant growth, whose purpose is to generate two identical daughter cells. Vegetative cell division, or mitosis, encompasses four sequential steps: two gap (G) phases separate the DNA replication (S phase) and chromosome segregation (M or mitosis). Determination of mitotic index (or cell division rate) in meristematic zones results very useful to know the health status and meristematic activity of the cells (Fiskesjö G. Hereditas 102:99–112, 1985). That is the main reason why this simple method has been widely used, especially when root growth inhibition is observed (Dayan FE, Romagni JG, Duke SO. J Chem Ecol 26(9):2079–2094, 2000), although as has been said, it can be also used to measure the mitotic activity of other organs. Mitotic index is used to measure cytotoxicity in living organisms (Smaka-Kincl V, Stegner P, Lovka M, Toman MJ. Mutat Res 368:171–179, 1996), based on the increase/decrease of the rate of cell division (Debnath B, Paul C, Debnath A, Saha D. J Med Plant Stud 4(3):107–110, 2016; Jain P, Singh P, Sharma HP. Int J Pharmacol Pharm Sci 3(2):46–52, 2016), and it can be simply calculated as reviewed in the present chapter.

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Graña, E. (2018). Mitotic Index. In: Sánchez-Moreiras, A., Reigosa, M. (eds) Advances in Plant Ecophysiology Techniques. Springer, Cham. https://doi.org/10.1007/978-3-319-93233-0_13

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A virtual laboratory on cell division using a publicly-available image database

Author(s): Eric A Shelden* 1 , Erika G Offerdahl 1 , Graham T Johnson 2

1. Washington State University 2. Allen Institute for Cell Science and University of California San Francisco

Published online: 28 Aug 2021

Keywords: image analysis microscopy mitosis Cellular Processes

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Potential video walkthrough spot

Cell division is a key concept in cell biology. While there are many popular activities to teach students the stages of mitosis, most make use of simple schematics, cartoons, or textbook diagrams. Others engage students in acting out the stages, or modeling them with physical objects (i.e. noodles, pipe cleaners). These approaches are useful for developing student knowledge and comprehension of the stages of cell division, but do not readily convey the real-life processes of mitosis. Moreover, they do not teach students how cell biologists study these processes, nor the difficulties with imaging real cells. Here, we provide an activity to reinforce student knowledge of mitosis, demonstrate how data on mitosis and other dynamic cellular processes can be collected, and introduce methods of data analysis for real cellular images using research-quality digital images from a free public database. This activity guides students through a virtual experiment that can be easily scaled for large introductory classes or low-resource settings. The activity focuses on experimentally determining the timing of the stages of cell division, directing the attention of students to the tasks that are completed at each stage and promoting understanding of the underlying mechanisms. Before the experiment, the students generate testable predictions for the relative amount of time each step of mitosis takes, provide a mechanistic reason for their prediction, and explain how they will test their predictions using imaging data. Students then identify the stages of cell division in a curated set of digital images and determine how to convert their data into relative amount of time for each phase of mitosis. Finally, students are asked to relate their findings to their original predictions, reinforcing their increasing understanding of the cell cycle. Students praised the practical application of their knowledge and development of image interpretation skills that would be used in a cell biology research setting.

Society Learning Goals

Cell biology.

• Cytoskeleton Structure and Function
• How do the different components of the cytoskeleton support a variety of cell functions, such as cell shape, division, movement, sensing the environment, and cell-cell communication?
• Cell Cycle and Cell Division
• How do cells conduct, coordinate, and regulate nuclear and cell division?
• Methods & Tools of Cell Biology
• How do the methods and tools of cell biology enable and limit our understanding of the cell?

Science Process Skills

• Process of Science
• Pose testable questions and hypotheses to address gaps in knowledge
• Interpret, evaluate, and draw conclusions from data
• Construct explanations and make evidence-based arguments about the natural world
• Modeling/ Developing and Using Models
• Build and evaluate models of biological systems
• Quantitative Reasoning/ Using Mathematics and Computational Thinking
• Apply the tools of graphing, statistics, and data science to analyze biological data
• Communication and Collaboration
• Share ideas, data, and findings with others clearly and accurately

Lesson Learning Goals

• Students will understand the events of mitosis and the cellular processes that occur during different stages of cell division.
• Students will understand how data on dynamic cellular events are collected and analyzed.
• Students will appreciate how cell biologists address experimental questions.

Lesson Learning Objectives

• Students will name and describe the salient features and cellular tasks for each stage of cell division.
• Students will predict the relative durations of the stages of cell division using prior knowledge and facts from assigned readings.
• Students will describe the relationship between duration of each stage of cell division and the frequency of cells present in each stage of cell division counted in a random sample of images of pluripotent stem cells.
• Students will identify the stages of cell division present in research-quality images of human pluripotent stem cells in various stages of cell division.
• Students will quantify, analyze and summarize data on the prevalence of cells at different stages of cell division in randomly sampled cell populations.
• Students will use data to reflect on and revise predictions.

Article Context

Article type, course level.

• Introductory
• Upper Level
• High School

Bloom's Cognitive Level

• Application & Analysis

Vision and Change Core Competencies

• Ability to apply the process of science
• Ability to use quantitative reasoning

Vision and Change Core Concepts

• Structure and Function
• Life Sciences Major
• Non-Life Sciences Major

Lesson Length

• Multiple class periods

Pedagogical Approaches

• Reflective Writing

Principles of How People Learn

• Motivates student to learn material
• Focuses student on the material to be learned
• Requires student to do the bulk of the work

Assessment Type

• Assessment of individual student performance
• Create a diagram, drawing, figure, etc.
• Create graph, table etc. to present data
• Design an experiment or research study
• Interpret data
• Written assignment: Essay
• Written assignment: Figure and or figure legend
• Written assignment: Lab report

INTRODUCTION

Cells are the fundamental unit of life and make up every living organism. All cells are produced by cell division, making the process one of the most important in all of biology. In addition to its role in reproduction, normal development and tissue maintenance, cell division underlies disease conditions, including cancer and hypertrophy. Not surprisingly, the mechanisms and control of cell division are central to the research efforts of many scientists and a description of cell division can be found in any biology textbook. The basic steps of cell division (prophase, prometaphase, metaphase, anaphase and telophase) are generally memorized by most biology students from textbook resources. With little first-hand experience in the underlying biology of these events, however, students ( 1 , 2 ) and even biology teachers ( 3 , 4 ) are known to struggle with memorizing and understanding the processes of cell division. Remarkably, problems associated with teaching cell division appeared in published literature well before the structure of DNA was established ( 5 ).

The teaching of concepts in cell division using texts, diagrams and models constructed from a variety of materials has been explored by numerous investigators ( 10 ) and offers students the opportunity to view stained chromosomes directly. Commercial and non-profit teaching kits for this activity are widely available. However, laboratory work with real biological samples is difficult to apply in introductory classes with large numbers of students, online teaching contexts, or settings with limited access to cell culture facilities and the necessary microscopes, cameras and computers ( 11 ). The complexities of working with real cells may limit the number of examples that students can view in a reasonable amount of time, reducing the potential for sampling cells in adequate numbers for statistical exploration. While plant cells are easy to obtain and manipulate, students may benefit from the opportunity to work with mammalian or even human cells, as they experience a more personal connection to the material. However, work with mammalian and human cells is technically difficult and involves specialized equipment needed to handle the exposure risks inherent to working with such cells. Moreover, current research in both academic and commercial settings is increasingly dependent on digital resources ( 12 ) and outsourcing of preparatory work to service laboratories ( 13 ). Thus, it can be argued that the use of digital images as the foundation of a data driven laboratory investigation offers potential insight into and training for real world research applications.

Analysis of digital images of cell populations containing dividing cells has the potential to address some of the limitations discussed above. The effectiveness of digital resources for teaching concepts in cell division has been previously assessed, and in general the available tools were shown to be as effective as hands-on laboratory exercises in promoting student understanding of key concepts. However, available digital and online resources are typically limited to small numbers of example images or videos ( 14 , 15 ). Recently, the Allen Institute for Cell Science ( allencell.org ) has provided public access to large sets of human cell images for research and educational purposes ( 16 ). The images were obtained by labeling human pluripotent stem cells with fluorescent markers for major cellular components including tubulin, the main building block of microtubules that form the mitotic spindle apparatus, and DNA. Images were obtained with high quality research-grade fluorescence microscopes at multiple image planes and are presented in an online viewer as both two- and three-dimensional data sets. Many thousands of examples are available (such as one shown in Figure 1). Some images represent individual cells suitable for high resolution structural analysis, while others show lower magnification fields containing twenty or more cells suitable for statistical analysis of cell populations. The image sets were obtained in a manner that permits rigorous scientific inquiry into cell morphology and intracellular organization and are therefore suitable for use in a hands-on virtual laboratory exercise that can provide insight into both the events of cell division and the sampling strategies that allow statistical analysis of dynamic cellular events. In addition to serving as a learning tool for the study of cell division, the activity can potentially provide students with exposure to a research method applicable to determining the mitotic index in a manner employed by scientists in both academic and commercial settings ( 17 , 18 ).

Figure 1. Example image of dividing cells obtained from the Allen Institute for Cell Science 3D Cell Viewer.

The activity described here was designed to improve engagement of students with the concepts of cell division by leveraging research resources provided by the Allen Institute for Cell Science. These are web-based resources, so this activity will require access to a computer with internet access. The activity is suitable for advanced undergraduate biology students but can be adapted for introductory biology for majors. This activity has been implemented as an extra credit assignment during an online web-based course as well as a take-home individual extra credit assignment in a face-to-face course. Potential adaptation for use as an in-class laboratory for individual students and small student groups is presented in the Discussion section.

In preparation for the activity, students should be provided with information about the stages of mitosis and the tasks performed by cells in each stage. They should be encouraged to consider the relationship between cellular tasks and duration of the mitotic stage. Instructors can present the material in the accompanying mini-lecture on cell division (Supporting File S1. A virtual laboratory on cell division-Mini-lecture) as well as the provided example images of cells at each stage of division (Supporting File S5. A virtual laboratory on cell vision-Example images of cells). The laboratory activity itself can be broken into two stages. In the first, students rationalize a prediction about the frequency of cells that will be present in each stage of mitosis in a random sample of pluripotent human stem cells. In Stage 2, students are provided with a random sample of real microscopic images of human pluripotent stem cells in various stages of the cell cycle. Students examine the images, quantify the number of cells in each stage of the cell cycle, and reflect on their predictions in light of individual and classroom aggregate data. Finally, students provide a written summary in which they consider how methods used to obtain, quantify and analyze data might influence the results obtained from their analysis. The activity was designed to promote both an understanding of mitosis and insight into the procedures used by scientists to obtain, quantify and analyze the behavior of cells.

INTENDED AUDIENCE

This activity has been used in an upper-level cell biology course at a large research-intensive university in both a face-to-face and an online course. Students in these courses include biology majors and first-year graduate students. The course is offered three times a year (fall, spring, and summer) with course enrollments ranging between 25 and 50. We anticipate this activity could be readily adapted for use in introductory biology classes in college and high school settings.

REQUIRED LEARNING TIME

The lesson takes ~2 hours to complete dispersed over two days of activity or instruction. The students spend ~30 minutes reviewing the events of cell division and producing a written hypothesis statement. Counting of the cells in images, tabulating, and summarizing results takes about an hour. Finally, producing a written summary and discussion takes about another 30 minutes.

PRE-REQUISITE STUDENT KNOWLEDGE

This lesson is intended for use after students have received instruction on mitosis. Students should be able to define the stages of mitosis and cellular structures that characterize the stages of cell division (e.g. mitotic spindle, microtubules, midbody, nuclear envelope). A good basic knowledge of cell division can be obtained by reading appropriate chapters in Alberts ( 19 ) , Lodish ( 20 ), Pollard ( 21 ), or an equivalent text. The Virtual Cell Animation collection has a helpful animation ( www.vcell.science ), and Scitable by Nature Education has a video of a mitotic cell ( https://www.nature.com/scitable/content/mitosis-6656772 ). The Allen Institute for Cell Science also has example images and videos of cell division at various magnifications ( 22 ), and a brief series of lecture slides describing stages of cell division with images from this source is included (Supporting File S1: A virtual laboratory on cell division-Mini-lecture). Prior to conducting their analysis of cell images, students may also benefit from gaining some experience with the controls of the 3D Cell Viewer ( 23 ). We have included a handout that provides an introduction to the laboratory, instructions for using the 3D cell viewer and suggestions for efficient counting of cells (Supporting File S2: A virtual laboratory on cell division-Handout, assignment and instructions), which could be provided to students either in its entirety or in sections. Finally, students should also understand how to tabulate and graph data using Excel or similar software.

PRE-REQUISITE TEACHER KNOWLEDGE

The teacher should be familiar with the stages of cell division and the underlying mechanisms and processes that occur in them. For example, in prophase the cell condenses its chromosomes, disassembles the interphase microtubule array and begins to assemble an early mitotic spindle. The cell also changes shape, often rounding up which collects the chromosomes and minimizes the space containing them. This in turn aids the cell in attaching chromatids to the mitotic spindle in prometaphase and reduces the potential of them to get lost in the cytoplasm, which leads to improper numbers of chromosomes in the two daughter cells (known as aneuploidy) and which can result in developmental defects or cancer. The breakdown of the nuclear envelope marks the end of prophase and the beginning of prometaphase. In prometaphase, chromosomes are captured by microtubules and attached to the mitotic spindle, which takes on its final shape. In metaphase, the cell checks to determine that all chromosomes have equal attachments to both poles of the mitotic spindle. This is the stage where an important cell cycle checkpoint must be passed for the cell to proceed through the remainder of cell division. Anaphase comes after metaphase and consists of the movement of chromosomes from the center of the cell to one mitotic spindle pole. This process is called "anaphase A". The spindle poles also move away from each other using microtubules to push the opposite pole away in a process called "anaphase B". Because of anaphase B movement, the mitotic spindle is usually much longer in anaphase than in metaphase. Finally, telophase is characterized by decondensation of chromosomes, the reassembly of interphase cytoskeletal arrays and reformation of the nuclear membrane. In many cells, a "midbody" comprised of bundled microtubules connects the two daughter cells for some time in telophase. Cytokinesis, the process that divides the cytoplasm of cells into the two daughter cells, usually occurs at the end of anaphase B or start of telophase. For many cells, prophase may be the longest stage of cell division, followed by metaphase and telophase, while prometaphase is very short and anaphase slightly longer, but shorter than metaphase or telophase ( 24 , 25 ). However, different cell types may display longer or shorter relative durations of these stages.

To assist students and prepare for the activity, instructors should have basic skills working with Excel or a similar spreadsheet program. Teachers may also wish to be familiar with the operation of the 3D Cell Viewer (Supporting File S2: A virtual laboratory on cell division-Handout, assignment and instructions) provided by the Allen Institute for Cell Science. This activity requires preparing Excel spreadsheets for each student or group of students with links to human cell images. Instructions for how to embed links to cell images within Excel spreadsheets are below in the section "Lesson Plan, Pre-lesson/lab preparations:".

SCIENTIFIC TEACHING THEMES

Active learning.

Students are actively engaged in the activity through analysis of authentic data and performing higher-order tasks (i.e. predicting, analyzing, evaluating, justifying). After reviewing the tasks that cells must achieve to complete cell division, students generate mechanistic hypotheses of how long a cell needs to accomplish each task and then predict the frequency of human pluripotent stem cells in each phase within their random sample of real images. Students analyze the real images of human pluripotent stem cells and directly observe the diversity of morphologies that real cells display. Finally, students aggregate observation data, reflect on hypotheses, and consider the impact of technical issues and sampling methods on data collection, quantitative results and the validity of their initial hypothesis.

Assessment of learning progress was measured by evaluation of two written documents generated during the first and second stage of the assignment. A possible grading rubric can be found below in the section "Lesson Plan, Grading rubric and notes". Emphasis was placed on understanding the process of obtaining data and the ability to support a line of reasoning with data obtained, rather than whether a student arrived at specific conclusions.

INCLUSIVE TEACHING

Our activity design contributes to inclusivity because it creates an environment where students' diverse backgrounds can be leveraged for learning. Specifically, students are encouraged to draw on their own existing cognitive resources (i.e. what they DO know) rather than engaging them from a deficit perspective by correcting or filling gaps in their knowledge (i.e. pointing out what students DON'T know). We further fostered inclusivity by using this activity as an alternative assessment (in contrast to high-stakes multiple choice or short answer exams) and incorporated multiple ways for students to demonstrate their understanding (e.g. written summary, group discussion). Our activity also requires students to use authentic data to answer authentic questions, and as such is a form of authentic assessment. Both alternative and authentic assessments have been identified as a best practice for inclusive teaching ( 26 ). Finally, this activity is beneficial to students with certain visual impairments because the images can be enlarged, and contrast can be enhanced.

LESSON PLAN

Pre-lesson/lab preparations:.

The emphasis of this lesson is to understand the tasks that a cell needs to perform during cell division, rather than just memorizing and identifying the names and characteristics of the stages. Before the lesson, the instructor prepares an Excel spreadsheet for each student or group of students containing links to the images of cells that will be used for analysis. We have provided several resources to facilitate this. An example student file is provided (Supporting File S3: A virtual laboratory on cell division-Example student Excel file) with links to 100 images, which can simply be duplicated for each student or group of students. Alternatively, individual links or groups of links can be copied from this file and pasted into separate Excel files. An additional file containing links to 927 3D image sets of cell groups stained for both DNA and microtubules available on the Allen Institute for Cell Science web site is also provided (Supporting File S4: A virtual laboratory on cell division-Complete Excel file). Instructors may wish to assign more or fewer images to each student or group depending on the availability of class time and the desired accuracy of the results. We suggest assigning 20 images which, depending on the number of cells in each image, has taken students between 20 and 40 minutes to analyze. We have included a file containing counts for total cells and dividing cells in 100 randomly selected links (Supporting File S6. A virtual laboratory on cell division-Example tallied results) sorted by the total number of cells in each image. Instructors may use this file to gain experience with counting and tallying the results of this analysis or to assign specific sets of images to students. For example, instructors may choose to assign images with low overall numbers of cells if class time is limited or assign sets of images that provide a known outcome. However, we believe the latter use should be adopted with caution and that the emphasis of this activity should be placed on the production of testable hypotheses and interpretation of obtained results, rather than that students obtain a specific result. Specifically, if small numbers of images are assigned for analysis to individual students, the probability that a small sample set will inaccurately represent the overall population behavior of cells is high. Instructors should also be aware that some students may find this file online through internet searches. Finally, we have created a java application available for download on sourceforge.net  or by request from the authors. The application accepts as input a text file containing names of students or groups of students, with one name per line. It then generates (within a designated folder) named Excel files containing a desired number of randomly selected links to images on the Allen Institute for Cell Science web site. Each link accesses one of the 3D image sets labeled for DNA and tubulin, but provisions are made that would allow an instructor to generate links to other data sets. The application was written to facilitate the application of this exercise to large classroom sizes where creation of individual Excel files by copying and pasting would be impractical.

IN CLASS OR AT HOME WORK:

This activity is broken into two parts: (1) generating a written hypothesis and (2) collecting and analyzing data followed by summarizing the results. An alternative approach would be to combine hypothesis development and data collection in one session with analysis and summarization of the data reserved for a second session or assigned as homework.

Students write down the events that occur at each stage of cell division. For example, a student may write that in prophase cells condense their chromosomes, disassemble the interphase microtubule array and begin construction of the mitotic spindle by separating their centrosomes. They then write a hypothesis regarding the length of time that cells will take to complete each stage of cell division and provide support for their hypothesis. For example, a student may hypothesize that telophase takes longer than anaphase and justify this statement by noting that anaphase only involves moving already attached chromosomes from the center of the cell to the spindle poles, while telophase involves assembly of the nuclear envelope, disassembly of the mitotic spindle, and relaxation of mitotic chromosomes. Based on their hypothesis, students predict what they will see in the real images of individual groups of cells in terms of frequencies of cells in each step of the cell cycle. For example, in a collection of photographs of a randomized population of dividing cells, longer stages of the cell cycle should be more common, while shorter stages should appear more rarely in the images. The students then submit their hypothesis and predictions for review. The instructor reviews the hypothesis and prediction; the hypothesis does not have to be correct. Students are asked to revise their statements if there is insufficient rationale supporting their hypothesis or if their predictions are not testable.

Once the instructor approves the hypothesis and prediction, students are provided an overview of the controls for the 3D Cell Viewer they will use to view images of cells on allencell.org (Supporting File S2. A virtual laboratory on cell division-Handout, assignment and instructions) and an Excel spreadsheet (Supporting File S3: A virtual laboratory on cell division-Example student Excel file). In the first column of the spreadsheet there are clickable links to images of randomly selected fields of cells within the 3D Cell Viewer. Clicking on each cell will open the corresponding image in the student's default web browser. Students count the total number of cells in each linked image. Then they count the number of cells in each stage of mitotic cell division within that image and enter these values into the Excel spreadsheet. In our classes, students were instructed to count cells in a minimum number of images (twenty), but students were offered HTML links to 100 different images.

Students may encounter difficulty in identifying prophase cells because nuclear morphology of DNA in non-dividing (interphase) cells is much more heterogeneous than usually depicted in textbook images. Additionally, students may have trouble distinguishing prometaphase cells from metaphase cells because the orientation of cells in a cell monolayer is not always parallel to the image plane. A set of example images for cells at each stage of division has been included (Supporting File S5: A virtual laboratory on cell vision-Example images) which may assist students with recognizing cells in various stages of division. The 3D Cell Viewer controls allow students to move the image plane through the cell in order to get a more accurate picture of the cell's morphology (Supporting File S2. A virtual laboratory on cell division-Handout, assignment and instructions). An option is also available that allows students to rotate the entire volume in three dimensions to better understand the structure of cells. Exposing students to the diversity of morphologies present in real cells in culture is a strength of this lesson.

After students finish counting and recording cells in the Excel spreadsheet, they summarize their data in a graph or chart. Students then evaluate the data in light of their initial hypothesis. Students are required to write down and turn in a report containing a summary of their data and a discussion of how the data support their hypothesis or what the discrepancies are between their hypothesis and their data and what the basis of these differences might be.

TEACHING PROMPTS:

Instructors may wish to provide students verbally or in writing with prompts to assist them in developing a written summary. Examples can include:

• Do your results support your hypothesis?
• If your results did not support your hypothesis, what are possible explanations for this?
• Which stages of cell division were most and least common?
• How does the number of cells you counted for a stage of cell division relate to the duration of that stage?
• Why do you think cells took so long to complete the longest stage of cell division?
• Why do you think cells needed so little time to complete the shortest stage of cell division?

Table 1. A Virtual Laboratory on Cell Division - Teaching Timeline

GRADING RUBRIC AND NOTES:

The lesson was implemented as a ten-point extra credit exercise with points awarded for both stages of the project. In stage 1, student hypotheses were examined to ascertain whether they were mechanistic and predictive. The emphasis was not placed on whether a student's hypothesis was "right", but rather on whether the student could present a logical explanation for their hypothesis and make logical predictions. Three points were awarded for completion of this part of the lesson. Students who turned in work before the deadline were offered the opportunity to revise their hypotheses without penalty if their hypotheses failed to include a mechanistic discussion. Examples of hypothesis statements that were given full credit would include statements such as "I think prophase will be the longest stage of cell division because the cell has to condense its chromosomes, assemble the mitotic spindle and disassemble the interphase microtubule network". Examples of statement that would be given partial credit or returned for revision would include " I think prophase will be the longest stage of cell division " (lacks mechanistic explanation) or " Prophase is the stage of cell division where chromosomes condense " (lacks prediction about the relative length of time a cell needs to accomplish the task).

Evaluation of the second part of the lesson consisted of examining whether the student had correctly identified cells at different stages of cell division, and whether they could provide a logical discussion of why the data supported or failed to support their initial hypothesis. In practice, the instructor examined whether students were able to correctly identify the stages of cell division in a small sample of evaluated images, using the individualized Excel files for reference. Seven of 10 points were awarded for this component of the lesson, with 3 points awarded for data analysis and 4 points awarded for the written summary. Similar to part 1, students that completed the work before the completion deadline were offered an opportunity to revise their discussions as needed. Students that had difficulty identifying specific stages of the cell cycle were also provided with additional examples or training and the opportunity to revise their summaries.

Possible grading rubric:

Hypothesis: (0-3 points)

• Clearly written, logical, focus on mechanisms and dynamics, duration of events of cell division, makes predictions, discussion of methods and potential impact on data, 3 points
• Clearly written, focused on events, makes predictions, states a hypothesis, 2-3 points
• Poorly written, disorganized, does not make predictions, 0-1 points.

Data analysis and presentation: (0-3 points)

• Presentation of results in a well formatted and labeled graph or chart that illustrates major findings, 3 points
• Presentation of results in a graph that illustrates major findings but missing labels, not well formatted, hard to read, 2 points
• Submission of a graph that does not illustrate major findings, graphs the wrong items, incorrect data, but good formatting, carefully and clearly labeled, 1-2 points
• Submission of a graph that does not illustrate major findings and poor formatting, 0 points.

Conclusion: (0-4 points)

• Thoughtful and logical conclusion, accurate summary of data, focus on mechanisms, dynamics, methods, discussion of hypothesis and why data did or did not support it, 4 points.
• Logical conclusion, accurate summary, some discussion of hypothesis and why data did or did not support it, 3 points
• Accurate summary of results, 2 points
• Some discussion of results, 1-2 points
• Conclusion lacks logic, little discussion of results, 0 points

TEACHING DISCUSSION

General observations.

To date, we have offered this laboratory to students in four classes ranging in size from 21 to 59 students. Sixty percent (77 total) students have voluntarily engaged in the activity, with participation ranging from 45 to 72 percent. Students generally found the activity interesting and beneficial. Students had some difficulty with determining the difference between interphase and prophase cells, in part because the nuclear morphology of interphase cells in the images is considerably more granular and heterogeneous than expected. Determination of the differences between prometaphase and metaphase cells is also potentially difficult, but observation of the mitotic spindle morphology by checking the box to display alpha tubulin may help with this determination. Due to the small number of images analyzed by each student (~20) and the random assignment of images, students occasionally produced results with significant over or under representation of specific stages of mitosis. Students often recognized the potential sources of this artifact or were guided to an understanding of the effects of small samples size during feedback on their written summaries. Given sufficient time, it may be useful for the instructor to have students combine their results to obtain larger sample sizes and more representative results.

EXAMPLE RESPONSES

As expected by most students, interphase cells made up the majority of cells in each image. Metaphase cells were the second most prevalent stage observed, followed by prometaphase and telophase. Prometaphase and anaphase cells were the rarest of observed stages. Examples of graphs submitted by students are shown in Figure 2.

Figure 2. Examples of results submitted by students after analyzing the number of cells in each stage of the cell cycle and cell division.

Most students made positive comments about the lesson. For example, one student wrote " Most importantly, the exercise allowed me to look at the theory presented in this last section from a practical perspective. I have been really struggling with the theory in this class and I think that such practical exercises with other cell processes would have helped me gain a much, much better grasp of the material presented." Students did express some frustration with identifying the different stages in these images. For example, a student wrote " Cells in interphase and prophase looked very similar to me ". It is likely that more training on the use of the 3D Cell Viewer and a larger number of example images would be helpful in addressing this problem. Our earliest implementation of this lesson used an image series that lacked alpha tubulin staining, however use of tubulin staining was added in a later version and facilitated identification of mitotic cells, especially those in prophase, which often displayed two separated microtubule organizing centers well before the appearance of a mitotic spindle. Telophase cells were also more easily defined by the presence of a mid-body of microtubules. In addition, students in face to face classes could benefit from the experience of the instructor and group discussion as they identify the stages of cell division. This is less easily implemented if the lesson is given as a take-home assignment and for online students. However, the difficulty in identifying images was also seen as a valuable experience and offered insight into the tasks faced by real biologists. For example, one response was " This assignment was useful in providing me with insight into cell biology methodology and data collection because it helped me to realize how difficult this research can be ."

POTENTIAL VARIATIONS

Although this activity was developed as a take home extra credit exercise for in class and distance learning students, it could readily be adapted to an in-class laboratory exercise for individual students and small student groups if suitable computer and internet resources were available. The two stages of the activity could be completed in two classroom sessions by some student groups, or two classroom sessions for introduction of cell division and discussion of results combined with out of class work assigned for cell counting online.

SUPPORTING MATERIALS

• S1. A virtual laboratory on cell division - Mini-lecture, containing diagrams of the stages of cell division, notes, and representative images of real cells at each stage from the Allen Institute for Cell Science web site.
• S2. A virtual laboratory on cell division - Handout, assignment and instructions.
• S3. A virtual laboratory on cell division - Example student Excel file, containing links to images on the Allen Institute for Cell Science web site.
• S4. A virtual laboratory on cell division - Complete HTML link list, containing 927 HTML links to all currently available images of cells labeled for DNA and tubulin on the Allen Institute for Cell Science web site.
• S5. A virtual laboratory on cell division - Example images of cells, containing images and notes for cells at each stage of cell division.
• S6. A virtual laboratory on cell division - Example tallied results

ACKNOWLEDGMENTS

This work was generously supported by the National Science Foundation (IOS 1457368 to EAS). We also thank the students in MBIOS401 at Washington State University for allowing us to use their responses to this exercise, and the Allen Institute for Cell Science founder, Paul G. Allen, for his vision and support.

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About the Authors

*Correspondence to:  School of Molecular Biosciences, Washington State University, Pullman WA 99164-7520  Email: [email protected]

Competing Interests

Kari Howey @ 10:12 am on 20 Feb 2023

Thanks so much! I really, really, really appreciate your work! Cheers!

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Esther Dalfo @ 1:35 am on 10 Aug 2022

Thank you very much for shareing this awesome tool!

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