The following points highlight the top three experiments on the living nature of protoplasm. The experiments are: 1. Demonstration of living nature of protoplasmic membrane 2. Demonstration on protoplasmic streaming 3. Demonstration of living nature of protoplasm by vital staining.
Experiment # 1
Demonstration of living nature of protoplasmic membrane:
(i) Effect of temperature on permeability:
Experiment:
A few Spirogyra filaments or Rhoeo peelings are mounted in water on a slide and one end of it is heated carefully over a flame. It is then examined under a microscope and any change in the appearance of the cell is noted. The filaments are dipped in water and examined again under the microscope.
Observation:
Shrinkage of protoplasm (plasmolysis) takes place. In case the cells are dead, no deplasmolysis takes place when dipped in water.
Inference:
Drastic plasmolysis takes place due to destruction of the living nature of plasma-membrane by high temperature treatment. There is no deplasmolysis and the cells do not revive when placed in water showing irreversible changes in the protoplasm and destruction of plasma-membrane brought about by high temperature.
(ii) Effect of high concentration of solutes on permeability:
Experiment:
Some Spirogyra filaments or Rhoeo peelings are kept in 2 M sucrose solution for about 15 minutes and then examined under the microscope. The tissue is then taken out and placed in distilled water for some time. This is again observed under the microscope.
Observation:
In a strong solution of sucrose cells show pronounced plasmolysis. When kept in water cells do not revive, i.e., there is no deplasmolysis.
Inference:
The strong solution of sucrose causes irreversible injury to the plasma-membrane and the protoplasm of cells leading to their destruction.
(iii) Effect of toxic substances on permeability:
Experiment:
Four skinned slices of beet root are washed thoroughly with water and dipped in four test tubes containing:
(a) 5% solution of H2SO4,
(b) 5% solution of CuSO2,
(c) 5% ethanol, and
(d) Distilled water and kept for about 40 minutes.
Observation:
The red anthocyanin pigment diffuses out in all the cases excepting in the tube containing distilled water.
Inference:
The semipermeable nature of the plasma-membrane is lost due.to treatments with different toxic substances and thus the colour diffuses out. In case of distilled water semipermeable nature is not affected and the colour does not come out.
Experiment # 2
Demonstration on protoplasmic streaming:
Experiment:
A portion of the peeling of freshly collected Vallisneria is mounted on a slide and is slightly, warmed. It is then examined under the high power of microscope.
Observation:
The rotatory movement of the chloroplastids along the boundary wall of the cell is observed.
Inference:
The protoplasm exhibits streaming movements particularly in plant cells with large vacuoles indicating its living nature. The exact cause of this movement’ is not yet clear. The exogenous supply of energy, however, enhances this rate and an un-coupler of oxidative phosphorylation like DNP (Dinitrophenol, 184-11 mg/litre) decreases this rate.
N.B. Experiments can be suitably performed using the cells of stamen hair of ebrina or Rhoeo and cells in the young tips or Chara or Nitella and the root cells of Limnobium spongia.
Experiment # 3
Demonstration of living nature of protoplasm by vital staining:
Experiment:
Some epidermal peelings of onion scales are immersed in a few ml of a 0.005% neutral red solution (dissolve 0.005gm neutral red in 100 ml 50% ethanol) in a watch glass. After 1, 5, 10 and 20 minutes interval peelings are taken out from the dye, rinsed in distilled water, mounted on slides and examined under the high power of microscope.
Observation:
Protoplasm of the cells is coloured red and the intensity of colour deepens with increase in time of treatment.
Inference:
Neutral red is a vital stain and all living protoplasm is stained by it. Prolonged treatment with the dye causes more adsorption of it on the protoplasm and the colour is intensified.