Staining is necessary to bring out clearly the constituent elements of the tissues and their relations with each other, and for the demonstration of histologic structures or chemical substances that would otherwise be nearly or wholly invisible. The technique of staining depends upon the fact that stains or dyes possess certain affinities for the tissue-elements or for certain simple or complex substances present in the tissues (microchemic reactions). These affinities vary greatly with the dye. Some dyes have an affinity only for single constituents of the tissue (elective or specific stains); others have an especial affinity for the nucleus (nuclear stains), others stain all the tissue-constituents diffusely (diffuse or protoplasmic stains). There are but few pure elective stains for single tissue-elements; the majority of stains will stain more than one of the tissue elements, but may show an especial affinity for certain ones. As a result of these variations in the affinities of dyes for the constituents of the tissues it becomes possible to manipulate the dyes or to combine them in such a way that a specific differentiation of many tissue-elements is possible through the use of different methods of staining. These methods are based in part upon the use of different mordants, the employment of several stains in combination or in succession, the mixture of stains to form a new staining compound, the phenomenon of metachromasia, the differentiation of certain tissue-elements by the removal of the stain from the structures for which it possesses a weaker affinity, and by the employment of different microchemic reactions. The two most commonly employed methods are the progressive, in which the stain is allowed to act until the affinities of certain tissue-elements have been satisfied when the staining process is interrupted; and the regressive, in which the tissue is over-stained, and the dye withdrawn from the tissue-elements for which it possesses the weakest affinities leaving the other elements stained. This latter process is usually called “differentiation,” and the chief substances used for such differentiating are dilute acid, acid alcohol, acid stains, aniline oil, aniline-xylol and alcohol. Some workers use the regressive method for such simple stains as hæmatoxylin, overstaining, and then differentiating with acid alcohol before counterstaining with eosin. The results obtained in this way are much less satisfactory than is possible with the progressive method.
Tissues may be stained in the body during life (intravital staining), or immediately after removal from the body (supravital or survival staining), either before or after sectioning. (See Page [217].) Fixed tissues may be stained in bulk or in sections.
Staining Tissues in Bulk.
This method is not often used in pathologic work. The fixed and hardened tissue is cut into small pieces, placed in the staining solution for several days, washed thoroughly, dehydrated in alcohol, imbedded, cut, and mounted without further staining. Alcoholic solutions penetrate best; hæmatoxylin, hæmalum, carmine and alcoholic solutions of the aniline stains may be used. Metallic impregnation (gold or silver salts) of fresh or fixed tissues is but little used in pathology. (See Staining of Nervous System, and Spirochætes.)
Staining of Sections.
Celloidin sections are lifted from water or alcohol into the stain by the needle or section-lifter. The use of the latter is advised, as by it the section can be floated flat on to the staining solution. When many celloidin sections are to be stained at once they can be stained in small tea-strainers and transferred in these from one solution to another. Paraffin sections may be floated directly on to the stain without removing the paraffin; or they may be stained on the slide or cover-glass after removing the paraffin, the stain being dropped on to the section, or the slide or cover-slip is immersed in the stain. Special staining-dishes for the staining of paraffin sections on slides and covers can be obtained. Paraffin sections transferred to celloidin sheets by the plate method can be put into the staining-solution while on the glass-plate, or the films can be detached and transferred from one solution to another by means of forceps. This is the easier way, and it is not necessary to touch the films with the fingers.
General Rules for Staining.
1. The stain should be filtered just before being used, in order to remove precipitates, moulds, etc. Unless they have been diluted most of them can be used over and over again, hence after using they should be filtered back into the stock bottle.
2. A liberal amount of stain should be used. Slides and cover-slips are given enough stain to cover completely the section, when the staining is done on the slide, or they may be immersed in staining-dishes. Plates and celloidin sheets should be stained in large trays. Sections and celloidin films should be flat without folds or wrinkles, and they should not touch one another when several are stained at the same time. Transference of the section from water or dilute alcoholic solutions to dilute or stronger alcohol respectively for a moment and then back again will usually straighten out curled or wrinkled celloidin sections.
3. Stain until the section is properly stained. Control this by removing it from the staining-solution and examining it in water on a glass-slide without a cover-slip, using the low-power. Sections will always appear more deeply-stained when cleared than when examined in water, hence due allowance should be made. Celloidin sheets can be examined on glass-plates. The time-limits given in staining methods are only approximate; no absolute rules can be laid down as to the length of time necessary to obtain a good stain. The methods of fixation and hardening, age of the tissue, age of the stain, etc., affect the staining power. Some stains lose their staining-power after a time; others require a period of ripening before they yield the best results. As a rule staining may be intensified or hastened by staining in the incubator or at a higher temperature, by concentrating the stain, or by the use of such substances as aniline oil. When differentiation is necessary the process should also be controlled by frequent examination of the section, as above for staining. Usually the section can be examined in the differentiating fluid.