Rates of penetration of fixing fluids.

Measurements of the rate of penetration of fixing fluids obviously provide an empirical basis for the minimum timing of tissue fixation, and pioneer studies to this end have appeared in a European literature: Tellyesniczky ('lo, '26), Underhill ('32), Medawar ('41), Baker ('58). In addition, however, experimental studies on fixing fluid penetration contribute information on how the fluids act and how cell appearances are changed by preservation. Tellyesniczky first showed that the components of a fixing mixture separate during penetration and that reagents penetrate tissue in sequence and at characteristic rates. A partially penetrated tissue shows a peripheral stratification with visibly different layers at varying depths from the surface. Measurements of slices cut at intervals from a chunk of tissue submerged in a fixing fluid provide data relating to the rate of penetration of the chemicals. The implication is clear, though it has not been stated unequivocally in the literature, that a cell at any locus within a tissue in a fixing mixture is provided with a succession of chemical environments. The various constituents of such a cell may be changed physically or chemically, or they may be inert to the first reagent that penetrates. The second reagent may react physically or chemically with the reaction products of the first reagent; it may react with previously unaffected constituents, or, again, it too may be inert. The same reaction possibilities are open to chemicals that penetrate later. The term reaction implies any type of physicochemical action: chemical combinations, responses to pH, denaturing, colloidal differences induced by cations, etc. It is unquestionably clear from the literature that each of these features may have an influence on the bulk, structure and appearance of cellular or tissue elements (Young, '35; Lassek, '50a and '51; Crawford and Barer, '51; Casselman, '55a; Freeman, Moyer and Lassek, '55; Bahr, Bloom and Johannisson, '58; Barrnett and Roth, '58; Hubendick and Blix, '57). One might expect, however, that a different catenation of physicochemical reaction might occur if the several reagents acted in some other sequence. This idea is no contradiction of the chemist's ideas on mass action; it merely suggests that the dropping of a tissue into a three-ingredient fixing mixture is really a complicated three-phase process in which the initial morphological and physicochemical state is potentially changed by the cumulative effects of primary, secondary and tertiary reactions. The probability is indeed small that a succession of chemical reactions, due to preservative chemicals penetrating in sequence, would kill or fix a cell and leave it exactly isomorphous and isometric with its living state. There is, however, a common idea, of obscure origin, that fixing mixtures are balanced media in which the swelling influence of one reagent is balanced by the shrinkage effects of another. This is obviously only a crude statement of the fact that mixtures even mixtures containing inert ingredients often produce less distortion, as seen with the microscope, than the ingredients of the mixture when used separately (Baker, '58). The notion of truly balanced media is contradicted by studies on fixed cells and by measurements of tissue volumes as modified by fixing fluids (King, '10; Tarkhan, '31; Ross, '53; Wiistenfeld, '55; Bloom and Friberg, '56; Bahr, Bloom and Friberg, '57). The functions of this communication are fourfold: (1) to provide data which will extend, substantiate or correct state-