The following points highlight the twelve experiments on stomata and transpiration.
They are: (1) Determination of Stomatal Frequency or Number of Stomata Per Unit Area of a Leaf (2) State of Opening of Stomata (3) Use of Darwins Porometer (4) Measurement of the Leaf-Area (5) Determination of the Total Number of Stomata in a Leaf (6) Determination of the Percentage of the Total Stomatal Area in Relation to the Area of the Whole Leaf
(7) Experiments with Cobalt Chloride Paper (8) Measurement of Rates of Transpiration (9) Measurement of Transpiration Rates—Quantitative (10) Determination of the Effect of Environmental Conditions on Transpiration Rates in Plants (11) Simultaneous Determination of the Amount of Water Absorbed and the Amount of Water Transpired by Plants and (12) Experiment to Demonstrate Suction or Pulling Force Developed due to Transpiration.
Experiment # I. Determination of Stomatal Frequency or Number of Stomata Per Unit Area of a Leaf:
Standardise an ocular micrometer with the help of a stage micrometer and calculate the value of 1o cular division. 1 mm=1000 µm; therefore 1 stage division is equivalent to 10µ. Then, if x stage divisions are equal to y ocular divisions, 1 ocular division is equal to 
µm. Standardise in both high and low powers of the microscope.
Now, find out the diameter of the field of vision of the microscope by the ocular
scale. The area of the field of vision is easily obtained. (The area of a circle is πr2 where r is the radius of circle, in this case half of the diameter of the field of vision.)
Count the number of stomata in this field. Take several readings by moving the epidermal peelings of leaves (e.g., Crinum) through the microscopic field of vision. The average of these readings divided by the area of the field is a measure of the stomatal frequency of the leaf, which is generally specific for each species of plant.
Experiment # II. State of Opening of Stomata:
(a) Lloyds method:
Peel off epidermal tissue from Rheo and other leaves and quickly put them into hot alcohol. The alcohol fixes the stomata, preventing any further movement of the guard cells.
Examine under microscope and measure:
(1) The entire area of stomatal apparatus—guard cells and the aperture and
(2) Actual area of the stomatal opening with the help of standardised ocular. (If both those structures are taken as roughly ellipsoidal in nature, the areas could be very approximately taken as equivalent to π(axb) where ‘a’ and ‘b’ represent half of the two axes of the ellipse.)
(b) Infiltration method:
Place a drop of absolute alcohol on the leaf and observe the rate of penetration, i.e., the rate of formation of transparent patches due to penetration of alcohol through the stomatal openings and its accumulation in the intercellular spaces.
(c) Durofix method:
Thinly smear both the surface of a suitable leaf while it is still attached to the plant carefully with Durofix adhesive. Allow the durofix to dry up quickly into a thin papery film. The film is now stripped off and the impressions of the stomata in the durofix-film can be observed under microscope.
Experiment # III. Use of Darwins Porometer:
Darwins porometer (Fig. 676) is a useful apparatus for following the changes in stomatal apertures, i.e., degree of opening of stomata. It essentially consists of a vertical tube, one end of which is dipped in a beaker of water.
The other end is fixed to a T-tube, into one arm of which is attached a rubber tubing provided with screw-cock and a small glass chamber (porometer cup) is attached with rubber tubing to the other arm.
The porometer cup is affixed to the undersurface of a dorsiventral leaf (stomata restricted only on the lower epidermis) and water is sucked up to a certain fixed level in the vertical tube and the screw-cock is closed and all connections made air tight.
The rate of fall of water in the vertical tube observed, provides a measure of the rate with which air passes out under suction through the stomatal openings of the leaf. If the cup is affixed to upper surface of such a leaf where there may not be any stomata, the water level in the vertical tube is maintained, but no fall in level is observed.
This certainly demonstrates that air cannot pass through the cuticle. The rate of fall of the water level and hence that of passage air is thus roughly indicative of the size of stomatal openings. If the rate of fall diminishes or becomes extremely slow, it evidently indicates that the stomata are closed or about to close.
Experiment # IV. Measurement of the Leaf-Area:
(a) The vast majority of leaves of oval, ovate and obovate shapes, which are the commonest in a tropical rain forest, have ratios very close to 2/3 of the overall rectangle. The area of a blade can thus be, more or less, correctly and at the same time quickly obtained, by multiplying the recorded rectangular area (length X breadth) by 2/3, over a wide range of leaf-blade forms.
(b) By graph paper:
Sketch the outlines of leaves on a graph paper and determine the area by counting the number of squares.
(c) By weighing:
Trace the outline of a leaf on a thick uniform cardboard. The leaf outline is cut out by scissors and weighed in a balance. Then, weigh a known area, say, about 4 sq. cm of the same cardboard.
The area of the leaf is thus easily found. The tracing of the outline of the leaf on cardboard paper is done in order to ensure uniform thickness of the area which is not obtained if actual leaves are used in weighing.
(d) By planimeter:
This is a simple gadget for determination of areas of unspecified surfaces. For routine work, this is a very useful apparatus as areas could be read out quickly from readings in a small vernier attached to the instrument.
Experiment # V. Determination of the Total Number of Stomata in a Leaf:
Find out the area of the field of vision of microscope under both powers and the average number of stomata present in this area. Area of the entire leaf is then determined by any of the four methods given above. The total number of stomata in the particular leaf is then easily obtained.
Experiment # VI. Determination of the Percentage of the Total Stomatal Area in Relation to the Area of the Whole Leaf:
The total number of stomata in the experimental leaf as also the area of a single stoma is found as in expts. IV and II (a) respectively. The product of these two values gives us the total area the stomata occupy in the leaf.
The area of the whole leaf is then determined as in expt. IV and the percentage the stomatal area in relation to the entire leaf is obtained. Stomatal area generally is only between 1 and 5% of the total area of the leaf.
Experiment # VII. Experiments with Cobalt Chloride Paper:
Soak filter paper in 5% solution of cobalt chloride and hang it up to dry. When dry, quickly cut into strips about 2.5 cm x 1.25 cm and dry these strips again thoroughly in an oven at a low temperature.
The original colour of the wet paper is pale pink, but when dried to a standard uniform shade, the colour becomes an intense blue. The uniform-sized dry strips are stored in a desiccator.
The treated blue dry paper is thus a moisture detector, turning pink when left in the air (the air containing moisture) or when placed in contact or near an evaporating surface. The time taken for a blue colour to disappear and attain a standard pink colour (determined by a stop watch) is a measure of the amount of evaporation as also the rates of water loss from the surface of the leaf to which it is attached.
The following tests can be conveniently performed with these dry cobalt chloride paper strips:
(a) Select a suitable leaf attached to a potted plant. Lay a piece of strip on each surface of a leaf and immediately press them gently down between two slides, held one above another with the help of clips (Fig. 677).
Compare the respective rates of water loss from both upper and lower surfaces of a leaf and also from young and old leaves.
(b) Compare the respective rates of one leaf kept in darkness for some hours (stomata closed) and the other kept in the open.
(c) Compare the respective rates of water loss from a leaf and also from an open water-evaporating surface (physical evaporation). For open evaporating surface a petri-dish containing water can be taken and the blue paper-strip is held by suitable arrangement as close to the water surface as possible, without actually touching it.
The time in seconds for a standard colour change of cobalt chloride paper over a free water-evaporating surface (s) divided by the time for the same colour change on the leaf (E) is a measure of what is sometimes defined as transpiration index- It is convenient to multiply this index by 100 to obtain the water loss from the leaf surface as a percentage of the evaporation from a free water surface.
Transpiration index S\E X 100.
Experiment # VIII. Measurement of Rates of Transpiration:
The rate of transpiration can be conveniently measured with the help of standard potometers, such as, Ganong’s and Farmer’s (Figs. 678 and 679). Ganong’s potometer essentially consists of a long narrow graduated tube held horizontally with two vertical short tubes, one of which takes a leafy twig (cut tinder water) through a cork while the other fitted with a stop-cock, acts as a reservoir.
The distal end of the horizontal tube is dipped into water in a beaker which may be coloured with eosin. The whole apparatus is filled with water while keeping the stop cock of the reservoir tube closed and all connections are made air tight with paraffin.
As water is lost by transpiration from the leafy twig, the coloured water is seen to enter horizontal arm from the beaker. The end of the tube dipping into water in the beaker is then momentarily removed from water to allow an air-bubble to enter it.
The tube is dipped again in water and as transpiration proceeds, the air-bubble is seen to travel with the transpiration current through the graduated arm towards the transpiring object. The time taken by the air-bubble to move through a specified length in the graduated tube may be noted.
If the volume of the tube is known, the rate of transpiration can be approximately calculated. When the bubble has nearly reached the reservoir tube, it can be pushed back into the water of the beaker by opening the stop-cock of the reservoir. A new bubble can again be introduced as before and the experiment repeated.
Farmer’s potometer (Fig. 679) is essentially a modification of Ganong’s.
Garreau,s potometer (Fig. 680) has been conveniently used for quantitative measurement of differential rates of transpiration from both upper and lower surfaces of a leaf. It consists of two small bell jars placed one above another in between which a leaf is placed while still attached to the potted plant as shown in Fig. 680.
At the narrow ends of the two bell jars, weighed amount of anhydrous CaCl2 is placed in two very small tubes. The broad rims of the bell jars in contact with the leaf surface are made air tight with vaseline.
At the two ends of the two bell jars, there are attached two oil manometers which ensure the maintenance of constant vapour pressure within the bell jars. A change in the vapour pressure within the bell jars shown by the manometers is indicative of the fact that either the connections are not air tight or that all the vapour given out by the leaf surface is not being absorbed by the CaCl2-tubes.
The whole arrangement is clamped vertically on a stand. After a few hours, the CaCl2-tubes are taken out and weighed again. The difference between the two weighings is a measure of the amount of water lost from the leaf surfaces. As the areas are the same, this is also a quantitative measure of the differential rates of transpiration from the two surfaces.
Experiment # IX. Measurement of Transpiration Rates—Quantitative:
Nearly fill a 500-ml conical flask with water. Insert a freshly cut (cut under water in order to maintain the continuity of water columns in the xylem vessels) sunflower or any other suitable leaf into the flask so that the petiole-end dips into the water.
Pour some olive oil carefully into the flask to seal the open water surface, thereby preventing evaporation of water. Weigh the whole arrangement quickly and allow it to stand in the open air or in a well-ventilated room.
After a few hours, weigh the whole arrangement again. Difference between the first and the second weights gives the amount of water transpired by the leaf. The water level of the flask is lowered, showing amount of water absorbed by the leaf.
Find out the area of the leaf by any of the methods given before and express your results as the amount of water lost per unit area of the leaf surface. Divided by the number of hours the experiment has continued, the rate of transpiration, per unit area per unit time, is obtained.
This simple arrangement of conical flask with water and oil on the surface and with the experimental leaf can be conveniently utilised for obtaining quantitative measurements of various correlation behaviours of transpiration rates such as with:
(a) Physical evaporation,
(b) Stomatal frequency,
(c) The degree of opening of stomatal aperture,
(d) The total area of the cross-section of xylem vessels in the petiole,
(e) The number of xylem vessels in the petiole, etc.
A quantitative idea of differential rates of transpiration from the two surfaces of the leaves can also be found out. (Three flasks are taken: (i) with untreated leaf, (ii) with leaf whose upper surface is thinly smeared with vaseline and (iii) lower surface smeared. The rates of transpiration in all three leaves are determined and differential rates from either surface are easily obtained by subtraction. This quantitative measurement can also be obtained, as indicated before, by Garreau’s potometer; a qualitative, as also roughly quantitative aspect, is given by cobalt chloride paper method.)
If a dorsiventral leaf with no stomata on the upper surface is selected, the rates of cuticular transpiration can also be determined by this method. In each case the area of the evaporating surface must be obtained and the results expressed as g per unit area per unit time.
If a quantitative estimate of amount of water lost per stoma in a leaf is desired, the total number of stomata in both upper and lower surfaces is determined, as also the total amount of water lost.
Amount of water lost from the transpiring surface divided by the number of stomata gives us an approximate measure of the amount of water given out per stoma. If comparative data are wanted for different plants, the transpiration rates per unit area divided by the number of stomata in that area, indicate a measure of rates of water loss per stoma.
Experiment # X. Determination of the Effect of Environmental Conditions on Transpiration Rates in Plants:
A few experiments can be devised to show that environmental conditions greatly influence transpiration rates in plants.
(a) Due to changes in the atmospheric humidity:
The same conical flask- oil-water-leaf arrangement can be conveniently utilised. The weighed control flask is kept in the ordinary air (relative low humidity).
The second weighed flask with leaf is kept under a bell jar. Keep a petri-dish with water inside the bell jar and the inside walls of the bell jar are covered with soaked blotting paper which ensures maintenance of almost saturated vapour pressure conditions (high humidity) inside the bell jar compared with the ordinary air outside.
Determine the areas of the two leaves and reweigh the conical flasks after the experimental period. Estimate the difference in the transpiration rates due to changes in the atmospheric humidity.
(b) Due to increase in the velocity of wind:
The control flask is kept in relatively still air in a well-ventilated room. The second flask is kept at a short distance from an electric table fan going at a moderate rate. After the experimental period, the transpiration rates are calculated due to increase in the wind velocities.
Experiment # XI. Simultaneous Determination of the Amount of Water Absorbed and the Amount of Water Transpired by Plants:
(a) The apparatus (Fig. 682) consists of a wide-mouthed bottle with an open-end graduated side-tube attached to it with an India-rubber cork. A small-rooted plant is introduced into the bottle through a split cork and all connections are made air tight.
The bottle is filled with water and the evaporation from the side-tube is prevented by pouring oil on the exposed water surface. The level of water in the side- tube is recorded. The whole arrangement is then weighed in a pan-balance and the weight noted.
After some time, the level of water in the side-tube falls, which is certainly a measure of the amount of water absorbed by the plant. The arrangement is reweighed and the difference between the first and the second weights evidently shows the amount of water transpired from the aerial leaf surface.
(b) The same relation can be experimentally determined by the previous conical flask-oil-water-leaf arrangement. Let the weight of the flask+water+oil (on the free water surface to prevent evaporation) =W1.
Flask + water+ oil+ leaf (cut under water) = W2.
After a desired number of hours, take again the following weights:
Flask + water + oil +leaf = W 3
Flask + water + oil only = W 4
Evidently W1 — W 4= amount of water absorbed by the leaf
W 2 — W 3 =amount of water transpired by the leaf.
Experiment # XII. Experiment to Demonstrate Suction or Pulling Force Developed due to Transpiration:
A glass-tube is taken and a leaf-shoot of Catharanthus (cut under water) is inserted into it with the help of rubber tubing and the connection made air tight with paraffin. The tube is now completely filled with water and holding it with a finger, the lower end is carefully dipped below the surface of mercury in a small vessel.
The whole arrangement is clamped vertically to a stand and is kept in bright sunlight in open air. Within a short time, the mercury i seen to rise in the tube which indicates the suction or the transpirational pull exerted on the water in the xylem vessels transmitted to the water column in the tube and ultimately to the mercury level in the beaker causing the mercury to rise in the tube (Fig. 683).
The weight of this transpirational pull can be easily calculated if the diameter of the tube is known and the height attained by the mercury column in the tube within a specified time is noted. The volume of mercury raised is evidently πr2h and specific gravity of mercury is 13-6. Thus the weight of the pulling force is πr2h X 13.6 g.
If comparative data for different species of plants are required then the total area of the transpiring surfaces (i.e., the leaves) of each must be determined by graph paper. The results then can be expressed as so many g per unit area of the aerial surface.
Experimental systems for demonstrating that both transpiration and the evaporation of water from a porous pot set up pulling forces which cause liquids such as water, mercury, etc., to ascend in the vertical glass-tubes, are shown in Fig. 684.
In the right- hand figure, instead of a leafy twig, there is a pot whose surface is dotted with minute pores. The pulling force or the tension developed due to both the processes is transmitted throughout the system, causing them to behave in a similar manner.