In observations of plasmolysis, plant cells are immersed in a hypertonic solution and examined with an optical microscope as water leaves the cells and the protoplast separates from the cell wall.
Common materials include Tradescantia, onion epidermis, and Elodea. Tradescantia epidermal cells are especially easy to observe because pigments in the cell sap make contraction of the protoplast relatively visible.
When a plant cell is placed in a solution more concentrated than its cell sap, water moves through the plasma membrane to the outside. The vacuole shrinks and the protoplast, including the plasma membrane, separates from the cell wall. This phenomenon is called plasmolysis.
If plasmolyzed cells are returned to water or a hypotonic solution, water may enter the cells again and the protoplast may return to contact with the cell wall. This is called deplasmolysis.
This article assumes an experiment in which plasmolysis of plant epidermal cells is observed using sucrose solutions of different concentrations, and presents the introduction, precautions, result items, example experimental values, calculations, sources of error, example results, and discussion examples.
Note:
This article is a study reference for discussing results obtained in biology experiments at schools and universities. The experimental conditions and observed values shown here are illustrative examples and are not actual results.
When using a razor, scalpel, forceps, slide glass, or coverslip, take care to avoid cuts and breakage.
Prolonged immersion in a highly concentrated solution may damage cells and make deplasmolysis difficult. Follow the laboratory manual and instructor directions during actual work.
Basics of Plasmolysis
Turgid State
When the external solution is less concentrated than the cell sap, water enters the cell and the vacuole expands. The protoplast is pressed against the cell wall and turgor pressure is high.
Incipient Plasmolysis
Water begins to move out of the cell and the plasma membrane separates from part of the cell wall. Separation may first be seen at corners of the cell.
Complete Plasmolysis
The protoplast contracts substantially and a clear space forms between it and the cell wall. The cell wall retains its shape while the protoplast becomes more rounded.
Deplasmolysis
When plasmolyzed cells are returned to water, water enters the cells and the vacuole and protoplast expand again. If the cells are not damaged, the protoplast comes back into contact with the cell wall.
Main Items to Record in the Results
- Observation material
- Tissue or region used
- Type of external solution
- Concentration of external solution
- Treatment time
- Observation temperature
- Eyepiece magnification
- Objective magnification
- Total magnification
- Number of cells observed
- Number of plasmolyzed cells
- Plasmolysis rate
- Whether plasmolysis was incipient or complete
- Position where the protoplast began to separate
- Space between the protoplast and cell wall
- Number of cells showing deplasmolysis
- Deplasmolysis rate
- Incipient plasmolysis concentration
- Cell damage or abnormal images
- Sketch or image of a representative observation
Example Experimental Conditions
| Item | Example condition |
|---|---|
| Observation material | Tradescantia epidermis |
| Treatment solution | Sucrose solution |
| Concentration | 0.00, 0.20, 0.30, 0.40, 0.50, 0.60 mol/L |
| Treatment time | 10 min |
| Observation temperature | 25 °C |
| Eyepiece | 10× |
| Objective | 40× |
| Total magnification | 400× |
| Cells observed | 100 at each concentration |
| Deplasmolysis treatment | After treatment with 0.60 mol/L, replace with water and observe for 10 min |
Example Experimental Values
Plasmolysis Rate by Concentration
| Sucrose concentration (mol/L) | Cells observed | Plasmolyzed cells | Plasmolysis rate (%) |
|---|---|---|---|
| 0.00 | 100 | 0 | 0 |
| 0.20 | 100 | 8 | 8 |
| 0.30 | 100 | 28 | 28 |
| 0.40 | 100 | 62 | 62 |
| 0.50 | 100 | 86 | 86 |
| 0.60 | 100 | 97 | 97 |
Degree of Plasmolysis
| Concentration (mol/L) | Not plasmolyzed | Incipient plasmolysis | Complete plasmolysis |
|---|---|---|---|
| 0.20 | 92 | 8 | 0 |
| 0.30 | 72 | 24 | 4 |
| 0.40 | 38 | 42 | 20 |
| 0.50 | 14 | 31 | 55 |
| 0.60 | 3 | 17 | 80 |
Example Deplasmolysis Observation
| Item | Number of cells |
|---|---|
| Cells plasmolyzed at 0.60 mol/L | 97 |
| Cells showing deplasmolysis after return to water | 84 |
| Cells that did not recover | 13 |
Characteristics of Observed Images
| Condition | Example observation |
|---|---|
| Water | The protoplast adhered to the cell wall and the vacuole appeared large |
| 0.30 mol/L | In some cells, the protoplast began separating at the corners |
| 0.40 mol/L | Plasmolysis was seen in more than half of the cells |
| 0.50 mol/L | Many cells showed substantial contraction of the protoplast |
| 0.60 mol/L | Complete plasmolysis was seen in almost all cells |
| Replaced with water | In many cells the protoplast again contacted the cell wall |
Calculation Methods
Plasmolysis Rate
If plasmolysis is observed in 62 of 100 cells at 0.40 mol/L, the calculation is as follows.
Plasmolysis rate (%) = Number of plasmolyzed cells ÷ Number of observed cells × 100
= 62 ÷ 100 × 100
= 62%
Complete Plasmolysis Rate
If 55 of 100 cells show complete plasmolysis at 0.50 mol/L, the calculation is as follows.
Complete plasmolysis rate (%) = Number of completely plasmolyzed cells ÷ Number of observed cells × 100
= 55 ÷ 100 × 100
= 55%
Deplasmolysis Rate
If 84 of 97 plasmolyzed cells recover, the calculation is as follows.
Deplasmolysis rate (%) = Number of recovered cells ÷ Number of plasmolyzed cells × 100
= 84 ÷ 97 × 100
≈ 86.6%
Estimating the Concentration for 50% Plasmolysis
If the plasmolysis rate is 28% at 0.30 mol/L and 62% at 0.40 mol/L, the concentration at which the rate reaches 50% can be estimated by linear interpolation.
Estimated concentration = 0.30 + {(50 − 28) ÷ (62 − 28)} × (0.40 − 0.30)
= 0.30 + (22 ÷ 34) × 0.10
≈ 0.365 mol/L
Therefore, the concentration at which 50% of cells are plasmolyzed is estimated to be about 0.37 mol/L.
Incipient Plasmolysis Concentration
Incipient plasmolysis refers to the state near the boundary at which plasmolysis begins. In experiments, the concentration at which about 50% of all cells show incipient plasmolysis is sometimes used as a practical criterion.
However, definitions and calculation methods differ among courses and laboratory manuals, so the criterion used should be stated explicitly.
Percentage of Cells That Did Not Recover
Non-recovery rate (%) = Number of cells that did not recover ÷ Number of plasmolyzed cells × 100
= 13 ÷ 97 × 100
≈ 13.4%
Main Sources of Error
| Source of error | Effect on observation | Likely tendency in results | Improvement |
|---|---|---|---|
| Thick epidermis | Cells overlap and are difficult to focus | Plasmolyzed cells may be missed | Collect a single thin epidermal layer |
| Sample drying | Water is lost before treatment | Overestimates plasmolysis rate | Mount immediately after collection |
| Coverslip pressure | Cells deform or are damaged | Damage may be confused with plasmolysis | Do not press strongly |
| Error preparing solution concentration | Actual concentration differs from target | Shifts estimated threshold concentration | Weigh accurately and make up to volume |
| Solutions mixed up | Concentration no longer matches observation | Concentration dependence becomes irregular | Label clearly |
| Incomplete solution replacement | Residual solution dilutes the new solution | Underestimates plasmolysis rate | Add from one side and absorb from the other |
| Different treatment times | Degree of equilibration differs | Comparisons among concentrations become inaccurate | Standardize treatment time |
| Delay before observation | Treatment lasts longer than planned | Overestimates plasmolysis rate | Record replacement time |
| Incorrect focus | Gap between membrane and wall is difficult to see | Incipient plasmolysis may be missed | Check multiple focal planes with fine focus |
| Differences in judging incipient plasmolysis | Classification differs among observers | 50% concentration shifts | Standardize criteria beforehand |
| Poor distinction between cell wall and protoplast | Boundary is misidentified | Plasmolysis rate becomes inaccurate | Compare with water-treated cells |
| Counting the same cell twice | Observed count becomes artificially high | Percentages become biased | Move the field in one fixed direction |
| Biased field selection | Areas with many plasmolyzed cells are preferentially chosen | Overestimates plasmolysis rate | Select randomly or at fixed intervals |
| Cell-to-cell variation | Cell sap concentrations differ | Responses vary at the same concentration | Observe many cells |
| Prolonged hypertonic treatment | Cells are irreversibly damaged | Deplasmolysis rate decreases | Do not extend treatment unnecessarily |
| Insufficient replacement with water | Hypertonic solution remains | Underestimates deplasmolysis rate | Replace with a sufficient amount of water |
Why the Shape of the Cell Wall Does Not Change
During plasmolysis, water is mainly lost from the protoplast and vacuole inside the plasma membrane. Because the cell wall is relatively rigid and retains its shape, the overall outline of the cell changes little even when the protoplast contracts.
Why the Protoplast Separates from the Cell Wall
In a hypertonic solution, water moves out of the cell and the vacuole shrinks. The volume of the protoplast consequently decreases, and the plasma membrane moves inward away from the cell wall.
Why the Degree of Plasmolysis Differs Among Cells
Even within the same tissue, cells differ in cell sap concentration, size, maturity, and degree of damage. Therefore, at the same external concentration, some cells begin plasmolysis while others do not.
Why Cells Recover When Returned to Water
When cells are returned to water, the solute concentration outside becomes lower and water moves into the cells. The vacuole expands again and the protoplast is pressed against the cell wall, bringing the cell closer to its original state.
Why Some Cells Do Not Recover
If hypertonic treatment is too strong or too long, the plasma membrane or protoplast may be damaged and may not return to its original structure even after water enters. Incomplete replacement with water can also prevent recovery.
Example Results Section
Tradescantia epidermal cells were treated for 10 min with sucrose solutions from 0.00 to 0.60 mol/L and observed for plasmolysis.
The plasmolysis rate was 8% at 0.20 mol/L, 28% at 0.30 mol/L, 62% at 0.40 mol/L, 86% at 0.50 mol/L, and 97% at 0.60 mol/L, increasing with sucrose concentration.
At 0.30 mol/L, some cells showed incipient plasmolysis, while at 0.50 mol/L and above many cells showed complete plasmolysis.
Linear interpolation between the values at 0.30 and 0.40 mol/L estimated the concentration for 50% plasmolysis to be about 0.37 mol/L.
When 97 cells plasmolyzed at 0.60 mol/L were returned to water, 84 recovered, giving a deplasmolysis rate of 86.6%.
Points to Connect to the Discussion
- What relationship was observed between solution concentration and plasmolysis rate?
- Why did the protoplast separate from the cell wall in a hypertonic solution?
- Why was the shape of the cell wall maintained?
- How were incipient and complete plasmolysis distinguished?
- Why did cells respond differently at the same concentration?
- How was the concentration for 50% plasmolysis determined?
- How is the incipient plasmolysis concentration related to cell sap concentration?
- Why did cells recover when returned to water?
- What may explain cells that did not recover?
- How did treatment time and solution replacement affect the results?
- How much could differences in observer judgment affect the results?
- How could the threshold concentration be determined more accurately?
Discussion Example
In this experiment, plasmolysis of Tradescantia epidermal cells was observed using sucrose solutions of different concentrations.
As sucrose concentration increased, the plasmolysis rate increased, reaching 8% at 0.20 mol/L, 62% at 0.40 mol/L, and 97% at 0.60 mol/L.
In highly concentrated external solutions, the water potential outside the cell is lower than inside the cell, so water is considered to have moved through the plasma membrane to the outside.
As water was lost, the volumes of the vacuole and protoplast decreased, and the protoplast including the plasma membrane separated from the cell wall. This was observed as plasmolysis.
In contrast, because the cell wall is relatively rigid, the outer shape was maintained even when the protoplast contracted. This demonstrates that the cell wall and plasma membrane are distinct structures.
Because plasmolysis was observed in 28% of cells at 0.30 mol/L and 62% at 0.40 mol/L, the concentration at which 50% of cells plasmolyzed was expected to lie between these values.
Linear interpolation estimated the 50% plasmolysis concentration to be about 0.365 mol/L. This concentration can serve as one indicator for estimating the cell sap concentration of the observed cell population.
However, not all cells have the same cell sap concentration, and cells also differ in maturity and size. Therefore, incipient plasmolysis did not occur simultaneously at a single concentration but increased gradually over a range of concentrations.
At 0.60 mol/L, many cells showed complete plasmolysis. A higher external concentration caused greater water loss from the cells, producing greater contraction of the vacuole and protoplast.
When cells were returned to water after plasmolysis, 84 of 97 cells recovered. This occurred because lowering the external solute concentration allowed water to re-enter the cells, expanding the vacuole and pressing the protoplast back against the cell wall.
Thirteen cells did not recover. Possible causes include damage to the plasma membrane or protoplast from high-concentration treatment, excessive treatment time, and incomplete replacement with water.
One source of error is judgment of incipient plasmolysis. In cells where the plasma membrane is only slightly separated from the cell wall, classification may differ depending on focus and observer.
If epidermal layers overlap or the focal plane is incorrect, the space between the plasma membrane and cell wall may be missed, leading to an underestimate of the plasmolysis rate.
If solution replacement is incomplete and much water remains on the slide, the actual sucrose concentration becomes lower than the set value. This can also produce a lower measured plasmolysis rate.
Conversely, if the sample dries before observation, water may already have been lost from the cells before treatment, causing an overestimate of the plasmolysis rate.
To improve the precision of the threshold concentration estimate, concentrations near 50%, such as 0.34, 0.36, and 0.38 mol/L, should be tested at smaller intervals using multiple specimens and many cells at each concentration.
In addition, observer differences can be reduced by defining a specific criterion for incipient plasmolysis, such as requiring a clearly visible region where the protoplast has separated from the cell wall.
Overall, the experiment confirmed that increasing the external solution concentration promotes water loss from plant cells and makes plasmolysis more likely. The recovery of many cells after return to water also showed that mild plasmolysis is a reversible phenomenon.
Discussion Example When Almost No Plasmolysis Was Observed
Possible reasons for observing almost no plasmolysis include a treatment solution that was too dilute, incomplete solution replacement causing dilution with water, an insufficient treatment time, or failure to detect incipient plasmolysis because the specimen was out of focus.
Discussion Example When Many Cells Plasmolyzed at Low Concentration
Possible reasons include drying of the sample, use of tissue with a low cell sap concentration, an error in preparing the treatment solution concentration, or counting damaged cells as plasmolyzed cells.
Discussion Example When the Deplasmolysis Rate Was Low
Possible reasons include excessive treatment time in the hypertonic solution, an excessively high concentration, damage to the plasma membrane, incomplete replacement with water, or drying of the sample during observation.
Discussion Example When the Relationship Between Concentration and Plasmolysis Rate Was Irregular
Possible reasons include mixing up solutions, differences in treatment time, differences in the sampled tissue region, too few observed cells, or inconsistent judgment criteria.
Summary
Plasmolysis is the phenomenon in which water moves out of a plant cell placed in a hypertonic solution and the protoplast separates from the cell wall.
As the external solution concentration increases, the plasmolysis rate rises and more cells show complete plasmolysis.
By determining the concentration at which 50% of cells are plasmolyzed, the incipient plasmolysis concentration can be estimated as an indicator of cell sap concentration.
When plasmolyzed cells are returned to water, they often recover, but cells damaged by strong or prolonged treatment may not.
In the discussion, it is important to relate the concentration dependence of plasmolysis, differences between the cell wall and plasma membrane, reversibility of deplasmolysis, treatment time, solution replacement, judgment criteria, and sample drying to the observed results and any deviation in the estimated concentration.
