Specific Study of Plant Tissues | Plant Anatomy

The following points highlight the three methods to make specific study of plant tissues. The methods are: 1. Preparation of Surfaces for Whole Mounts 2. Section Cutting 3. Maceration Method. 

Method # 1. Preparation of Surfaces for Whole Mounts:

For the preparation of surfaces of different plant organs such as leaves and stems various techniques are followed. These preparations actually represent tangential sections of the plant materials, but are much better in quality and firmness.

Leaf Surfaces:

The epidermis of many leaves can be readily removed by peeling it off the fresh leaf. This can be done by folding the leaf to break the surface and then, by holding with forceps as thin a layer as possible, peel it back. Alternatively, the broken surface can be stripped directly, by pulling one part of the leaf downward relative to the other. This is called the peeling method.

The second method is known as the scraping method. Both fresh and fixed leaves can be used. Fixed leaves should be washed in water. Cut out a suitable piece from the leaf. Place the surface for study face down on a glass plate. Put a few drops of sodium hypochlorite or some domestic bleach on it.

Hold one end of the leaf segment tightly with a cork and scrape the other end gently with a safety razor blade. Hold the blade at 90° to the leaf. Continue scraping, adding more sodium hypochlorite, if necessary, till a thin and clear area is obtained.

Cut off this thin area and brush off loosely adhering cells (Fig. 2.1). Observe the piece under microscope after mounting it in water to see if scraping is complete. Place the surface the right way up before final mounting after staining.

Stem Surfaces:

The scraping method can be applied to obtain a thin and clear stem surface. Divide the stem segment into two longitudinal halves and use one half.

Cuticular Preparations:

The cuticle can be separated from the leaf by using various chemicals. One method is to digest away the leaf tissues with nitric acid. The cuticle often floats to the surface or, after thoroughly washing the leaf, the cuticle can be teased out from the dissolving tissues.

Surface Replicas:

A good imprint of the leaf surface can be obtained in the following way — wipe the leaf surface with acetone to clean it. Apply a thin film of cellulose acetate on it. Apply over it nail varnish with a brush. Several applications may be necessary. Allow it to dry. Remove the dried film of nail varnish and mount it in a medium with a different refractive index to observe it clearly under microscope.

Method # 2. Section Cutting:

Free-Hand Sections:

For general class work free-hand sections are usually made. The material is held by the thumb and forefinger of the left hand. A drop of water is put on it. An unused safety razor blade is held by the thumb and forefinger on one of its blunt edges (shorter edges).

A drop of water is placed on the near sharp edge of the blade. The material and the blade are allowed to meet at right angles to each other. A section is cut with one stroke. The stroke is directed from near the body to away from it. A section should be cut with one stroke.

While cutting sections the blade is placed on the forefinger of the left hand and the thumb is held at a slightly lower level so that it is not injured by the blade. A number of sections are cut and then transferred to a watch glass containing water. Sections which appear to be relatively thinner — and so, more transparent — are mounted on a slide in water and in a row.

These are examined under the low power of the microscope and a few sections are selected for detailed observation and staining. With a little practice sections can be obtained which are thin enough for study under magnifications of 100 to 400 X or even more. A difficulty which is often encountered is that the sections often become oblique.

To avoid this, the blade and the material should always be held at right angles and the section must be cut with one stroke. Both the material and the blade should be kept sufficiently moist to avoid drying and, conse­quently, air-clogging of the sections. Sections can also be cut with a razor. Such a razor must be of high quality and it should be regularly polished and sharpened.

Microtome Sections:

Extremely thin and high quality sections are obtained with a rotary microtome. The details of microtome sectioning have been discussed under Cytology portion. It is a laborious and time-consuming process but very effective. While studying development anatomy, it is a must.

Wood Microtome Sections:

The hardness of wood makes its sectioning very difficult with razor blades. A wood microtome (sledge or sliding microtome) is suited for this purpose.

Sections of 15-30 urn thickness can be cut with this microtome which are suitable for histological studies. The material is firmly held in a universal clamp (to allow for correct orientation and the knife is brought towards and over the material). Water or 50% alcohol is used for lubrication.

Hand Microtome:

It consists of a flat, circular, metal or glass-topped metal plate with a hole at its centre. The plate is rigidly attached to a hollow cylindrical handle through which runs a plunger that has a screw attached to it.

A tissue core is taken with a cork borer and inserted into the hollow cylindrical handle. Sections of 20 nm or thicker are cut by drawing a razor or a safety razor blade across the plate, each time readjusting the specimen height by advancing the screw.

Softening of Woody Tissues:

Hard materials, such as wood, are cut into 1 cm cubes. These are boiled in water until they become waterlogged, i.e., they can now sink in cold water. These are now curled, clamped and sectioned. If boiling cannot make them sufficiently soft, they need a jet of steam directed on the surface to be cut (Fig. 2.2).

Use of Pith and Cork:

Many plant materials, such as leaves and soft stems, are too delicate to be held firmly and sectioned. In such cases pith (potato tuber and carrot root) or cork can be used. In case of pith, take a rectangular block of potato tuber or carrot root, avoiding the skin.

Make a partial longitudinal slit in its middle region, leaving the lower part intact. Insert a piece of the material to be sectioned into the slit and cut sections of both pith and material. In case of cork, select a suitable bottle cork having very few lenticels (Fig. 2.3).

Cut out a circular slice. Cut this disc of cork across the diameter and place the two halves side by side. For transverse sections of stems, the cork or pith (Fig. 2.3) will help to keep the material correctly oriented, without allowing it to be excessively compressed.

For making longitudinal sections, the cork or pith slice is cut across the diameter at an angle (Fig. 2.3). This helps to keep the material in proper position while sectioning.

In case of leaves, scooping out of pith or cork tissue is not necessary. A suitable piece of leaf can be inserted within the pith or cork and then sectioned. Wide leaves can be folded several times to form a sandwich in the cork or pith (Fig. 2.4). If the leaves are narrow, several of them held in a bunch —just like the pages of a book — can be inserted within the cork or pith.

Very soft hydrophyte stems and leaves sometimes pose problems. The thin cell walls burst easily if compressed when turgid. One way to overcome this is to allow the material to go limp on the bench for 15 minutes to ½ hour, and then section with the help of pith or cork.

Types of Sections:

Stems are normally sectioned in the middle of the internode or at any other convenient level. Leaves are normally sectioned across its broadest region, i.e., half-way along with length of the lamina. For detailed studies and to study the development of organs, such as root-stem transition, root and shoot apices, leaf and branch traces and gaps, etc., serial microtome sections are needed.

When a section is cut at right angle to the long axis of the organ, it is transverse section (T.S.).

If the section is cut parallel to the long axis of the organ, it is called longitudinal section (L.S.).

Longitudinal sections are of two types. If the plane of longitudinal section is parallel to the radius of the organ, it is called radial longitudinal section (R.L.S.).

If the plane of longitudinal section is parallel to the circumference of the organ, i.e., at right angles to the radius, it is called tangential longitudinal section (T.L.S.). T.L.S. and R.L.S. are particularly important while studying wood anatomy (Fig. 2.5).

Method # 3. Maceration Method:

Sections of plant organs do not give an accurate conception of the real nature of the cells which make the organs. It gives only a section view of the cells where the distribution of the tissues is better understood. The cells cannot be studied in their entirety. The method which reveals the cells in their entirety is the Maceration Method.

In maceration the plant tissue is treated with chemicals which dissolve the middle lamellae and also some or most of the cells with thin cellulose walls and allow the remaining cells to become separated from one another.

Consequently, the cells can be viewed in their entirety and the nature of cell wall thickening including pores and other distinguishing features are clearly brought out. The technique of maceration including the nature of the chemicals used depends mainly on the hardness of the tissues to be macerated.

Jeffrey’s Method:

Cut the material (dry or fresh) into small slices about 0.5 mm thick. Boil and cool repeatedly until they become free of air. Macerate in a solution of equal parts of 10% nitric acid and 10% chromic acid. The material with the solution may be heated in a paraffin bath for woody tissues but not for soft and herba­ceous tissues.

The duration of treatment depends on the hardness of the tissue, but cells begin to separate in about 24 hours’ time. A glass rod with rounded end may be used to tap the material gently in order to loosen the cells. If the material does not crumble easily, change the macerating fluid and continue treat­ment. Wash thoroughly in water to remove the acids.

Usually, the macerated tissue along with the fluid mixture is poured into a large volume of water and the macerated bits are picked up with a brush. A centrifuge may be used to separate the macerated tissues, if it is available. The material is now stained with any suitable stain, such as eosin or water soluble safranin, mounted in 50% glycerine and observed.

For permanent staining, leave the macerated tissue in 1% safranin for 6 hours, rinse thoroughly in water and then dehydrate by rapid addition of hygrobutol. Give two changes of pure hygrobutol and then add a little balsam highly diluted with hygrobutol. Evaporate it down to a mounting consistency. Mount this on slides.

Quick Methods of Maceration:

Jeffrey’s method, although quite adequate for all kinds of tissues, is a very slow method.

There are other methods which are much quicker:

1. Herbaceous materials, such as Cucurbita stems are cut into thin slices and taken in a test tube along with a little 10% or 20% KOH solution.

Hold the test tube with a test tube holder and boil it over a burner keeping the mouth of the test tube away from your body. Shake the tube frequently to avoid bumping of the material. Add more KOH solution, if necessary. When the tissue comes out in shreds, wash and stain as before.

2. Both woody and herbaceous materials can be macerated in a mixture of 10% nitric acid and 10% chromic acid taken in equal proportions. Cut the materials into thin slices and take them in a test tube along with some macerating fluid. Boil as before, taking greater precaution to avoid bumping. AVOID INHALING THE VAPOUR as nitric acid vapour is highly toxic. Wash and stain as before.

3. Very hard, woody materials are macerated with conc. nitric acid along with a pinch of potassium chlorate (KClO₃). Cut the materials into thin bits and take them in a test tube along with a little conc. nitric acid. Add a pinch of KClO₃ and boil taking utmost care.

Nitric acid reacts with KCIO₃ to produce potas­sium perchlorate (KClO₄) which is highly reactive and toxic. It should never be allowed to bump and the fumes must not be inhaled. Always keep the mouth of the test tube directed away from your body.

Shake the tube regularly. Take it away from the burner frequently. Minor accidents often occur due to negli­gence. Maceration takes place very quickly and the entire tissue dissolves, if it is slightly overdone. Wash the tissue immediately when maceration appears to be still incomplete. Stain as before.