Examination of Tissues

The methods of tissue examination may vary according to the structural and chemical components of the cells to be studied, the nature and amount of the tissue to be evaluated and the need for an immediate examination of a tissue structure.

Examination may be done on fresh or preserved tissues, depending upon necessity. Fresh tissues have the advantage of being examined in the living state, thereby allowing protoplasmic activities such as motion, mitosis, phagocytosis and pinocytosis to be observed. Its use has been limited, however, because of the fact that tissues examined in the fresh state are not permanent, and therefore, are liable to develop to develop the changes that have usually been observed after death.

Methods of fresh tissue examination

1. Teasing or Dissociation – is a process whereby a selected tissue specimen is immersed in a watch glass containing isotonic salt solution, carefully dissected or separated and examined under the microscope, either unstained, by Phase Contrast or Bright Field Microscopy, or stained with differential dye.

2. Squash preparation (Crushing) – is a process whereby small pieces of tissues, not more than one mm in diameter, are placed in a microscopic slide and forcibly compressed with another slide or with a coverglass.

If necessary, a vital stain may be placed at the junction of the slide and the coverglass and allowed to be absorbed by the tissue through capillary attraction.

3. Smear preparation – is the process of examining sections or sediments, whereby cellular materials are spread lightly over a slide by means of a wire loop or applicator or by making an opposition smear with another slide. This technique is especially useful in cytological examination, particularly for cancer diagnosis.

a. Streaking – with an applicator stick or platinum loop, the material is rapidly and gently applied in a direct or zigzag line throughout the slide, attempting to obtain a relatively uniform distribution of secretion. Too thin or thick smear have to be avoided since they make the tissues unsuitable for examination.

b. Spreading – a selected portion of the material is transferred to a clean slide and gently spread moderately thick film by teasing the mucous strands apart with applicator stick.

This method is a little more tedious than streaking, but has the advantage of maintaining cellular interrelationships of the material to be examined. It is especially recommended for smear preparations of fresh sputum and bronchial aspirates and also for thick, mucoid secretions.

c. Pull–apart – this is done by placing a drop of secretion or sediment upon one side and facing it to another clean slide. The material disperses evenly over the surface of the two slides in the opposite directions may be necessary to initiate the flow of materials. The two slides are then pulled apart with a single uninterrupted motion, and the specimen placed under the microscope for immediate examination, or applied with vital stain.

This is useful for preparing smears of thick secretions such as serous fluids, concentrated sputum, enzymatic lavage samples from the gastro–intestinal tract and blood smears.

d. Touch preparation (impression smears) – is a special method of smear preparation whereby the surface of a freshly cut piece of tissue is brought into contact and pressed on the surface of a clean glass slide allowing the cells to be transferred directly to the slide for examination by Phase Contrast Microscopy or after vital staining.

It has an added advantage in that the cells may be examined without destroying their actual intercellular relationship, and without separating them from their normal surrounding.

4. Frozen section – this method is normally utilized when a rapid diagnosis of the tissue in question is required and especially recommended when lipids and nervous tissue elements are to be demonstrated.

Very thin slices around 10 – 15u in thickness are cut from a fresh tissue frozen on a microtome with CO₂ or on a Cyrostat, a cold chamber kept at an atmospheric temperature of –10^oC to –20^oC, transferred to a dish containing isotonic salt solution and stained for microscopic examination.

Processing of Tissues

Fresh tissues are usually examined when there is an immediate need for evaluation. A better and more effective means, however, of studying tissues, whether normal or abnormal, is by examination of their section and smears which have been permanently preserved, stained for demonstration of specific structures, and mounted on a glass slide with coverslip for permanent keeping.

Solid structures and tissues must be preserved and carefully processed in the following order:

1.    Fixation

2.    Dehydration

3.    Clearing

4.    Infiltration

5.    Embedding

6.    Trimming

7.    Section – cutting

8.    Staining

9.    Mounting

10.  Labeling