Preparation for microscopic study
Objects to be studied under a microscope need preparation. If the object is small enough, through light can pass, and the study in finest details is not required a whole mount preparation will be sufficient. In case of large objects they are to be cut in thin sections for the study. This is called histological preparation.
A. Whole Mount Preparation
Killing and fixing
In case of live specimens they are to be killed. Killing must be instantaneous. This is done with chloform, ether, acetone, alcohol, formalin, menthol, Bouin’s fluid etc. These chemicals serve as fixatives also. Specimens contracting or retracting suddenly in contact with chemicals are narcotised with menthol, thymol, poisonous gas etc. and then killed.
Staining is the act of giving colour to something. The killed or preserved specimens are thoroughly washed with water. This is followed by staining. In case of aqueous dyes, the specimens are first stained and then dehydrated. Dehydration means removal of water. In the use of alcoholic dyes the staining is done after dehydration to that concentration in which the dye is dissolved.
The period required to stain a specimen depends both on the size of the object and concentration of the dye in the solution.
Over staining, if any, is corrected by treatment with extremely diluted acid solution in water or alcohol according to the case, for a short period followed by immediate washing with water or alcohol. The water is removed from the specimen in steps by passing it through 50%, 70%, 90% and 100% alcohol.
The dehydrated specimen is cleared in clove oil. The oil is washed in xylol and the specimen is mounted on a slide in canada balsam or DPX, and covered with a cover slip. Grooved slides are used for thick specimens.
Specimens do not need dehydration or cleaning. Mount in water or saline solution or in 50% glycerine. If required the cover slip is sealed with bees wax or nail polish. A turn table is very convenient for the purpose.
B. Histological Preparation
Collection of tissue
For sectioning, tissues are to be collected from live specimens. In small vertebrates, the animal is paralysed by damaging the brain. In larger vertebrates, the same result is achieved by striking the head against a hard object. In still large species the tissues are collected immediately after killing the animal for some other purpose or a small piece of tissue for some other purpose or a small piece of tissue is cut by surgical operation. In invertebrates, the tissues are usually collected from live specimens without such pretreatment.
Killing the tissue without any structural or chemical change is known as fixing.
In practice, it has not yet been possible to prepare a fixative which satisfies all the requirements fully.
The tissues are cut into small pieces, washed with physiological solution, if required, immersed in the fixative and left there for a definite period. The common fixatives used are Bouin’s fluid, Zenker’s fluid, Carnoy, Carnoy-Labrum etc. The specimen tubes with the fixative and tissues should be subjected to a vacuum pump for a few minutes to remove any air in the tissue which may enter during collection. Air interferes with the processing. The time required for fixation varies with the tissue.
This is usually done with water till the reagent is completely removed. If the per cent of water in the fixative is below 50, it should be washed only in solution recommended for the purpose.
Carry the tissues through grades of alcohol, viz. 50%, 70%, 90% and 100%, the period of treatment with each grade depending on the size of the tissue. Give two changes in absolute alcohol.
Transfer the tissue to ceder wood oil. Within a few days the tissues turn transparent and are ready for infiltration.
This is to replace oil with paraffin. The intercellular spaces are completely occupied by paraffin.
The tissue is washed in xylol and transferred to a covered porcelain crucible containing xylol and molten paraffin at a ratio 1:1 and the crucible is put in a temperature controlled paraffin bath for 10 to 15 minutes. After the period, the tissue is transferred to another covered crucible containing molten paraffin only, kept in the same bath. The time required for infiltration varies with tissues. Usually infiltration is completed within 90-120 minutes. After the desired period paraffin blocks are made with the tissues.
Paraffins of different melting points are used in different seasons.
This is done with a microtome. Sections for routine work are cut 5-6 micrometer thick. A grease free slide is taken. One side of the slide is marked with the help of a diamond pencil. A droplet of Mayer’s albumen is smeared on the marked surface. A thin film of water is put on the slide and the paraffin ribbon with sections are put on it. The slide is heated on a hot plate and the sections are properly stretched. The water is drained off and and the slide is dried on a hot plate.