Experiment on Protoplasm and Plasma Membrane | Botany

Here is an experiment to demonstrate the living nature of protoplasm and plasma membrane.

All living organisms are composed of the essential living substance, ‘Protoplasm’, which is a jelly-like colloidal substance confined within small chambers called ‘Cells’. The protoplasm undergoes a series of chemical changes that regulates the entire cellular activity.

The major characteristic features of living system is expressed by protoplasm and its components viz. movement, permeability, metabolic reactions etc. Temperature, toxic chemicals, conc, solution etc. have profound effect on the permeability of plasma membrane, thereby leading to plasmolysis of cells.

The streaming movement of protoplasm or response to vital stains by protoplasm also indicate its living nature. The thermal death point of cells and antagonism of ions in living system also provide support to the living state of protoplasm and its components.

The following experiments are designed to demonstrate the living nature of protoplasm and its associated cellular components:

(a) Experimental studies on protoplasmic movement

(b) Effect of temperature, toxic chemicals and conc. solutions on protoplasmic membrane permeability

(c) Experimental demonstration of living nature of protoplasm by vital staining

(d) Determination of thermal death point of cells

(e) Demonstration of ionic antagonism in living tissue systems

Experiment:

(A) Protoplasmic Streaming (Fig 3.8):

A portion of a freshly collected young leaf of Vallisneria is mounted on a slide in water and then examined microscopically.

Then a few drops of hot water are put on the mounted strip and re-examined after 3-5 minutes.

Finally a few drops of ice-cold water are poured on the mounted leaf strip and examined after 3-5 minutes.

(B) Membrane Permeability:

(i) Effect of Temperature:

A few Spirogyra filaments or pealing’s of Rhoeo leaves are mounted in water on a slide and then examined under microscope. Then a few drops of hot water are added to the mounted specimen.

(ii) Effect of Concentrated Solution:

Some Spirogyra filaments or Rhoeo leaf pealing’s are kept in 1M sucrose solution for about 1-5 minutes and then examined under the microscope. Then the treated tissue is taken out and placed in distilled water for some time and examined microscopically.The rotatory movement of the chloroplasts along the boundary wall of the cell is observed.

(iii) Effect of Toxic Substances:

Slices of beetroot are taken and washed in distilled water and then placed in test tubes containing one of the following solutions:

5% soln. of H₂SO₄

5% soln. of CuSO₄

5% Ethanol

Distilled water

Then after 30 minutes the tubes are examined.

(C) Vital Staining of Protoplasm:

Some epidermal pealing’s of onion scales are immersed in a few ml of 0.05% neutral red solution in a watch glass. After 10-15 minutes, the pealing’s are taken out, rinsed in distilled water, mounted on a slide and examined under microscope.

Observations:

The rotatory movement of the chloroplasts along the boundary wall of the cell is observed.

The rate of movement of chloroplasts in the protoplasm is enhanced.

The protoplasmic movement has almost ceased.

Shrinkage of protoplasm (plasmolysis) takes place. Deplasmolysis may take place on dipping the treated cells in cold water after a while. If not, then the cells are dead.

In concentrated solution of sucrose, cells show marked plasmolysis. When kept in water, cells do not show deplasmolysis.

The red betacyanin pigment diffuses out in all the cases except the tube containing distilled water.

Protoplasm of the cells is coloured red.

Inference:

The protoplasmic streaming move­ment is indicating its living nature.

The exogenous supply of energy enhances the rate of movement.

Plasmolysis takes place due to destruction of the living nature of plasma membrane by heat. If there is no deplasmolysis of cells on dipping in cold water, then living nature of the membrane and protoplasm is permanently destroyed, i.e. the cells are dead.

The concentrated solution of sucrose causes irreversible injury to the plasma membrane and the proto­plasm of cells leading to their destruction.

The membrane permeability is lost totally due to the effect of different toxic substances. As such, the pigment is then leached into the solution.

Neutral red is a vital stain and all living protoplasm is stained by it.

(D) Thermal Death Point of Plasma Membrane:

Materials and Equipment’s:

1. Beetroot.

2. Beaker, thread, stand, scalpel, dist. water, burner, tripod stand, thermometer, etc.

Procedure:

1. Cut a block of Beetroot and wash away the exuding pigments by distilled water thor­oughly.

2. Tie the block with a thread and suspend it in a beaker (500 ml) containing distilled water. The beaker is placed on a tripod stand (Fig. 3.9).

3. Note the initial temperature of water and heat the beaker on a burner.

4. Record the change in temperature of water inside the beaker and gently stir it by a glass rod.

5. Carefully note the temperature at which leach­ing of pigment from the beet root block starts. This can be done more accurately if the bea­ker is placed against a piece of white paper.

Observation:

Record the water temperature at which leaching of pigment starts.

Inference:

The leaching of pigment is due to the loss of permeability of cell membrane due to higher tempera­ture. This particular temperature is considered as the thermal death point of membrane and cell too. Repeat the experiment 3 times to get a more accurate result.

(E) Ionic Antagonism on Membrane Permeability:

Experiment:

Two stock solutions of NaCI and CaCL₂ of conc. 1.88% (which is more or less isotonic to cell sap) are prepared.

Six petridishes are taken, each having one of the following solutions:

(a) Distilled water —9 ml

(b) NaCI: CaCI₂ -9:0

(c) NaCI: CaCI₂ – 4.5 : 4.5

(d) NaCI: CaCI₂ -6:3

(e) NaCI: CaCI₂ -3:6

(f) NaCI: CaCI₂ – 0.9

Equal slices of Beetroot of uniform thickness are cut with the help of a cork borer and they are thoroughly washed in distilled water to remove the adhering pigment from the wounded cells.

About 4-5 such slices are then immersed in each petridish containing the above solutions in aforementioned proportions. Observations are made at intervals of 5 minutes for 1/2 hr to note whether any red colour diffuses into the external solutions.

Observations:

Diffusion of red colour occurs first in petridish (b) then in (c) and last of all in (a). No colour diffusion takes place in case of petridish (d), (e) and (f).

Inference:

NaCI increases the permeability of the membrane to a point which is injurious to cells but CaCI₂ acts in reverse way, i.e., it decreases permeability to a point which is also detrimental. When two salt solutions are mixed in a proper ratio, their combined effect neutralizes the injurious effect on permeability. This is called ionic antagonism which is only seen in a living system.

N.B.:

Detection of colour diffusion can be made more accurately if the peri-dishes are placed on white paper.