Question

what is the tissue processing ? please write long

Answer 1

TISSUE PROCESSING

Tissue processing is concerned with the diffusion of various substances into and out of stabilized porous tissues.The rate of solution diffusion through tissues is proportional to the concentration gradient (Fick's law). Impregnation of the material resists the impact of cutting action of knife. Paraffin wax is the substance commonly used for this purpose. Wax is prefered to other substances because it readily passes from solid to liquid state at temperatures that do no spoil the material.

Preparation of sections by paraffin impregnation method involves many complex methods.

1. Fixation

2. Dehydration

3. Clearing

4. Infiltration

5. Casting of wax impregnated material into blocks

6. Fixing of paraffin block to the block holder of a microtome

7. Cutting of serial sections

8. Affixing the ribbon in well arranged rows on glass slides

9. Removal of wax

10. Staining and mounting

Fixation

Fixation is the preservation of the form, shape, chemical make up and structure of cells in their original state. Fixatives are the combination of more than one chemical compound. The general principal involved in the combination of reagents to form a fixing fluid is the maintenance of a balance between the properties of the different reagents involved. Fixation is usually done in two ways; chemical agents (chemical fixation) and by sudden freezing (freeze fixation).The commonly used fixatives are ethyl alcohol, formalin, acetic acid.

Dehydration

Dehydration is the removal of all traces of water from cellls and tissues with the help of chemical agents. Dehydration consists of treating the material with a series of solutions containing progressively decreasing concentrations of water and a progressively increasing concentration of the dehydrating agent. The interval in each grade must be determined by the size, nature of the material and the solubility of the residual reagents left in the material.

Clearing

When the materials are dehydrated in reagents that are not miscible with the wax used in paraffin method of preparing serial sections, or with the resinous media in which sections are permanently mounted.

Infiltration

This is the process of dissolving paraffin in the solvent that contain tissues. In this process the concentrationn of paraffin gradually increases and the quantity of the solvent gradually decreases. Toward the end of the process, the solvent is completely eliminated from the system.

The paraffin used for infiltration must have a constant melting point, appropriate hardness and smooth texture and must be free particles of dirt, water and volatile or oily components. Paraffin of melting points between 50-55 C is ideal for botanical work.

Casting of wax impregnated material into blocks

The technique of enclosing of the impregnated tissue in a block of solidified paraffin is termed embedding. For embedding, pour the molten paraffin into paper boat to fill the three forth of it. Wait for a few seconds untill a thin skin forms on the surface. By this time a layer of hardened wax might have been formed at the bottom of the boat. Then the tissues are then transfered to the boat. Pour more molten paraffin to fill the boat up to its brim, if required. Warmed needles are used to orient the tissue in the appropriate position. Wait till the wax is sufficiently soldified. Blow on the surface untill the paraffin becomes sufficiently firm. Then pick up the boat and immerse it in the cold water in a tray, keeping a heavly scalpel over it. Then the paper boat is taken out, the paper is peeled off, and the paraffin block is stored dry in properly labeled packets.

Fixing the paraffin block to the holder of a microtome

The block is divided into separate pieces, each piece containing the tissue to be sectioned. After deciding which face of the tissue is to be cut the piece is trimmed roughly into a rectangular prism. Block is placed on a block holder  and fixed it using a heated scalpel and molten wax. The holder with block is cooled under water. Wipe off water and neatly trim the block with a razor blade. The face across which the cuts are to be made should have its upper and lower sides parallel.

Sectioning

By setting appropriate thickness thin sections are made using microtome. The mictrotome is used in moderate speed such that there is no compression of sections and successive sections adhere to form a ribbon. The ribbon is held by camel hair brush and collected on a clean paper.

Affixing the ribbon

The ribbons is cut into convienient lenghts and placed on the slide. The slide is then warmed gently untill the sections flatten out and expand without melting. The slide is marked and left for drying.

Staining and mounting

First step is the removal of paraffin from sections. The slide carrying serial sections is dropped into a couplin jar containing xylene or benzene to remove wax. Transfer the slide now to a mixture of absolute alcohol and xylene, then to absolute alcohol, and finally to water. After removal of every trace of wax , the sections are stained in desired combinations of stains. After staining slides are passed through dehyrants and brought to xylene. Place a thin coat of Canada balsam (an adhesive) on the top of slide and leave the solvent to evaporate from this. A clean cover slip is passed over the flame and pressed over the slide, the slide is left to dry.

Cleaning and labeling

When the slides are completely dried, the surface balsam should be removed with a blunt knife. Write the serial number of the slide .