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PREPARATIONOF SLIDES

Slide making is an important part of many areas of biological, medical,veterinary and forensic sciences and you will often be required to prepare different kinds of slides in-house. Specimens may be smears of fluids, thin sections or whole mounts of all or part of an organ or organisms. In all cases the material is mounted on a glass slide prior to its examination.

Two main types of preparation are used:

(1) Temporary, and

(2) Permanent.

Temporary preparations are examined and then discarded, usually on the same day; whereas permanent preparations may remain in good condition for years.

The techniques involved in preparation are described here.

Temporary Preparations

These preparations may be needed for a matter of minutes or hours only. They are mounted in water or dilute 1,2,3propanetriol(glycerol) or other fluid of low volatility. After examination they are discarded.

The material under examination can be fixed and stained or even examined in a living state, for example protozoa such as amoeba. In this case a harmless aqueous stain such as I% methylene blue can be used -it would then be known as a ‘vital stain’.

If it is necessary to keep the slide for a matter of hours it is possible to reduce evaporative losses from the edges of the coverslip by painting a ring of gum or molten wax or nail polish around its edge.

It is always desirable to use a cover-glass over your temporary preparation. If it is omitted, the curvature of the drop of liquid in which the object is mounted causes optical distortions. An additional problem is the danger of contaminating your microscope’s objective lenses.

The preparation of stained specimens for microscopical study either temporary or permanent generally involves the following three processes (additional processes may be used in making permanent preparations).

(1) Fixation

For fresh tissues, the main aim of fixation is to kill tissues rapidly by

precipitating proteins. This minimizes post-mortem changes. The reagent used is called a fixative, the most commonly used being 70% alcohol. Othercommon fixatives are Bouin fluid and formalin. Different fixative are used depending on the nature of the tissue whether soft or hard.

Tissues should be washed well after fixation, using the same solvent as the stain, in order to remove all traces of the fixative. If this is

not done, tissues may not stain properly and some types of fixative may crystallize out (fixation is not necessary if the material is already preserved).

(2) Staining

The object of staining is to accentuate the distinction between the different components of a tissue or organ.

(3) Mounting

Mounting media employed for temporary preparations include water and 1,2,3-propanetriol (glycerol) 30%-50% aqueous solution.

Permanent Preparations

If the slide is to be kept for long-term reference, for days or even years, it mustbe made as a permanent preparation. This is achieved by:

(1) Dehydrating the specimen after staining (usually with a graded series of alcohols);

(2) Clearing it -treating it with a solvent that is mutually soluble between alcohol and the mounting medium [traditionally clove oil or 1-2dimethylbenzene (xylene)]; and

(3) Mounting it under a cover glass in a preservative which dries hard and has a refractive index similar to glass, e.g. Canada balsam or Euparal or DPX mountant which gets dried up easily as opposed to Canada balsam which takes longer time.

The making of a permanent stained preparation mounted in Canada balsam involves five processes:

(1) Fixation,

(2) Staining,

(3) Dehydration,

(4) Clearing, and

(5) Mounting.

Fixation and staining have already been mentioned in the earlier sub-section and the purpose of dehydration, clearing and permanent mounting is outlined below.

(1) Dehydration

The purpose of dehydration, i.e. the removal of water, is to allow complete infiltration of tissues with Canada balsam. Unless all traces of water are removed, infiltration is incomplete, the tissues appear opaque and bacteria ldecay ultimately sets in.

If carried out too rapidly, dehydration causes distortion and shrinkage, especially of delicate tissues, by setting up violent diffusion currents. It should therefore be done gradually and sufficient time allowed for the complete extraction of water. Dehydration is commonly effected by the passage of the stained specimen or slide through successively stronger solutions of ethanol

(ethyl alcohol) ending with immersion in absolute alcohol 100% ethanol (2-changes).

CAUTION: Alcohol is a highly flammable material. Adequate ventilation and no naked flames are essential safely precautions.

• Clearing

Where the dehydrating agent is immiscible with the mounting medium, it is necessary to introduce an intermediate fluid that is miscible with both. Such a fluid is known as a clearing agent.

The main purpose of clearing is to remove all traces of alcohol, thus allowing the tissues to be infiltrated with the Canada balsam or other mountant.

Examples of clearing agents are 1,2-dimethylbenzene (xylene) for small soft tissues; clove oil and cedar-wood oil for thick tissue. Incomplete dehydration is indicated by cloudiness in theclearing agent and the slide should be returned to absolute alcohol. Toluene is another good clearing agent but it is a bit costly.

2. Mounting

Permanent preparations are obtained by enclosing tissues in solid, resiniferous media such as Canada balsam1DPX. After the tissues have been cleared, they are mounted in a semi-fluid 1,2-dimethylbenzene(xylene) balsam mixture. The1,2-dimethylbenzene (xylene) subsequently evaporates and the balsam hardens.

However, there are some disadvantages of using this mountant.Xylene is inflammable and toxic; the drying process is prolonged and thismountant discolours with time. It is advantageous to use DPX because it dries up easily. To hasten the process of drying up, slides may be kept in oven at lower temperature for sometime.

Use of stains

A great variety of stains are in common use. In all cases the aim is to give different parts of the specimen different colours and colour density and hence increase optical contrast between them. Two different staining methods are in common usage.

(1) Progressive staining: This involves leaving a tissue in the stain until nuclei, for example, are deeply stained and cytoplasm is only faintly stained. It is advisable to dilute the stain beforehand.

(2) Retrogressive (regressive) staining: This entails deliberate, over-staining followed by destaining which results in differentiation. Best results are obtained when overstaining is prolonged. This method depends on differential rates of stain extraction. For example, acid alcohol removes haematoxylin (haematoxylin is a nuclear stem that stains only the nucleus) more rapidly from the cytoplasm than the nuclei.

Other important aspects of staining procedures are as follows.

(1) Stains may be used singly or in combination i.e. counterstaining.

(2) The use of two stains together is known as double staining.

(3) Stains used in combination should be complementary in colour, e.g. light green and safranin (green and red) used in plant tissue staining.

Stains are either general or specific. A general stain, e.g. borax carmine, stains all parts of a tissue usually in different densities or shades of the same colour.

A specific stain acts on one or more components of a tissue or organ in a selective manner, e.g. safranin stains lignified plant cell walls red.