In order to know how the different reagents used in cytopreparatory techniques react with the two main components of the cell (the nucleus and the cyto-plasm), it is necessary to know their respective compositions.
Since the ground protoplasm of most cells is amphoteric in reaction, it can in principle react with both acid and basic dyes. It is possible to shift the existing balance of charges in the cytoplasm-proteins by changing the pH of the dye solution. Several staining methods are based on this pos-sibility, especially those where quantitative measurement of protein is required.
Although a knowledge of histopathology is of paramount importance in diagnostic cytology (Koss, 1979), it is far too simple to state that the appearance of cells in a cytological specimen is identical to that of the corresponding cells in histological material. There are several reasons for the presence of differences in cell images in his-tology and cytology, and of differences in cell harvest in the two methods.
The cytologist is primarily concerned with the overall picture of the cell, showing nucleus and cytoplasm and, if possible, staining the nucleolus differentially. Often a distinction between nucleus and cytoplasm will be sufficient; in other cases a more detailed study of the cell's components is called for. So, in the first place, specific nuclear and cytoplasmic stains are needed (see chapters 5 and 6). Furthermore it may be important to study the specific constitution of the nucleus, i.e. its DNA and RNA content, or of the cytoplasm, i.e. protein, RNA, lipid and glycogen contents. In all cases it will be necessary to know something about stains and the mechanism of staining.
In certain situations the cytologist wants to have a first impression of the cell sample taken, or a preliminary diagnosis is desired by the patient and/or clinician. The first is important in aspiration cytology, to check whether the lesion or organ is aspirated and whether the sample contains diag-nostic cells. A preliminary diagnosis of an aspirate is necessary in order to plan further investigation or an operation. In the latter circumstance, it takes the place, or is applied in conjunction with, frozen-section histology (Pickren and Burke, 1963). In both instances, it is important that the cell sample be ready for microscopy soon after it is taken. Thus short staining and, if fixing is needed, short fixing times are extremely important. Since a refined diagnosis is not needed here, a compromise in visual quality, in order to meet the desired time limits, is acceptable.
In section 1.1.4 we saw that the nucleolus comprises a fibrillar and a granular zone, each containing RNA and ribonucleoproteins. There is some DNA present in the fibrillar (inner) zone. The ribonu-cleoproteins are non-histones, which means they are acid. Both Papanicolaou's and Romanowsky— Giemsa's staining methods show the presence, form and number of nucleoli quite clearly. In Papan-icolaou's method they are either red or blue (depen-ding on the pH of the fixative and of the staining baths). In Romanowsky—Giemsa's they are all shades of blue.
This chapter is the 'cookery book' part of this volume. First, it will give practical information on how to process cytological material from various sites; how to preserve and fix cells; how to make cytological slides; how to process difficult-to-han-dle material; how to combine the various fixing and staining methods; and how to prepare material for adjuvant scanning and transmission electron microscopy.
So far we have looked at the presence and structure of cell components (chapter 1), the structure of colouring agents (sections 3.3 and 4) and the ways of linking the two (section 3.7). If staining, and thus visualising, cells and their components could be based on the simple linking of colouring agents to cell components in cells that in all aspects are identical to those in the original sample, then it would be reasonable to assume that all cell com-ponents would still be present, that the colouring agents would be able to enter the cell, and that they could easily reach those cell components with which they are best suited to form a link. As far as the last of these is concerned, it would be necessary for the substrate not to be obscured by any other cell component.
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