A Silver-Schiff Reaction for Cytological Studies: Light Microscope Observations by JOHN H

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A silver-Schiff reaction for cytological studies: light microscope observations By JOHN H. D. BRYAN (From the Department of Zoology and Entomology, Iowa State University, Ames, Iowa, U.S.A.) With 1 plate (fig. 1) Summary A method is presented in which a complex metal-ion (ammoniacal silver) is substituted for leuco-basic fuchsin in the well-known Schiff reaction for aldehydes. The reagent is reduced to metallic silver by tissue aldehydes and submicroscopic particles of the metal are deposited at the reactive sites. When the reagent is used in the Feulgen reaction chromosomes may be 'stained' in a specific manner to show the localization of deoxyribonucleic acid. For such a purpose, fixation should be carried out with acetic acid and alcohol mixtures rather than with solutions containing formalin or dichromate; these latter substances preserve more of the tissue lipids and thus complicate the issue by producing cytoplasmic staining (plasmal reaction). Introduction THE possibility of using specific metallic 'stains' to visualize cell structures such as chromosomes at the electron microscope level has long been a subject of interest. In particular, a reagent specific for DNA has been much sought after. The possibility of using metal-ion substitutes in the well-known Schiff reaction has attracted attention since such a method should give very precise localization of DNA strands. Ammoniacal silver nitrate is well known as a reagent for the demonstration of aldehydes (the so-called silver mirror test). Silver-Feulgen procedures, in which a complex silver ion is substituted for the leuco-basic dye reagent (Schiff's base) have been introduced by Bretschneider (1949); Bradfield (1954), and Jurand and others (1959). How- ever, these methods have not been widely applied. The foregoing procedures were applied directly at the electron microscope level with little or no mention of parallel studies at the light microscope level. It occurred to me that a preferable procedure would be to investigate the use of such reagents with the light microscope and then, after suitable exploration of variables and further refinement of the method, to pursue the most promising technique at the electron microscope level. The present report is based on light microscope investigations. Electron optical studies now in progress will be reported separately.

Material and methods The silver reagent was made up by titrating dilute silver nitrate with dilute ammonium hydroxide until the precipitate which formed was just redissolved. The exact concentrations of the reagents are not critical provided that they [Quart. J. micr. Sci., Vol. 105, pt. 3, pp. 363-366, 1964.] 364 Bryan—A silver-Schiff reaction are close to 2%. I have found that ammoniacal silver solutions of higher concentration (5-10%) are subject to more rapid deterioration during the staining period. Insect tissues (Orthoptera and Diptera) containing relatively large chromosomes were chosen for the initial experiments. Gonads were fixed in acetic- alcohol, washed with alcohol and soaked in 45 % acetic acid for a few min. Fragments of tissue were then squashed in 45 % acetic acid and the coverslips removed by a dry ice procedure (Conger and Fairchild, 1953). Salivary glands were dissected free of fat bodies in a drop of insect Ringer, squashed in 45 % acetic acid and the coverslips removed as before. Slides were processed directly or, in the case of salivary squashes, following a short fixation in acetic-alcohol. After removal of the coverslip and after any post-squashing fixation slides were washed in alcohol, overcoated with collodion and run down to distilled water. The slides were then hydrolysed in iN HC1 at 6o° C for 12 min and washed in distilled water until free of chloride. Staining in the silver-Schiff reagent was carried out for 6 to 8 hours at 40° C. Follow- ing staining, slides were washed in several changes of distilled water and immersed in a dilute solution of ammonium hydroxide for 2 to 5 min. After rinsing again in distilled water, slides were dehydrated in the usual manner and mounted with resin media following removal of the collodion film. Un- hydrolysed slides served as controls as in the usual Feulgen procedure. Similar slides were stained by the usual Feulgen procedure and served as reference standards. The silver-Schiff reagent is not very stable and each batch should be discarded after 2 days. Also a note of caution should be appended. This ammoniacal solution is liable, upon prolonged standing, to produce black needle-shaped crystals of highly explosive silver 'fulminate' (nitride?) (Mellor, 1939; Smith, 1943). After use, therefore, the staining solution should be discarded promptly. Observations The reaction results in the deposition of metallic silver particles of submicroscopic dimensions at the site of free aldehyde groups. Chromatin is coloured in a range from golden brown to black depending upon the concentration of DNA. Thus interphase nuclei are lowest in visual contrast and condensed mitotic chromosomes, and needle-shaped sperm, highest. Comparison of the test slides with unhydrolysed controls indicated that the reaction was specific for aldehyde groups unmasked by acid hydrolysis (no structural details could be seen in the controls except with the aid of phase- contrast optics). Photomicrographs of Feulgen and silver-Schiff preparations are shown in % i- Discussion There are certain side-reactions which take place during staining. The most troublesome of these, in the present work, has been the non-specific J. H. D. BRYAN Bryan—A silver-Schiff reaction 365 deposition of silver oxide. Initial experiments involved the use of 10% (w/v) silver nitrate solution and staining at 6o° C for several h or overnight. This procedure resulted in a very heavy deposition of silver oxide. Overcoating slides with collodion gave improved preparations in that less oxide was seen in the tissue. Reduction of the strength of the reagents and of the staining temperature together with the retention of the collodionization step further improved the results. Residual oxide could be easily removed from slides processed in this way by immersion in dilute ammonium hydroxide. Staining can be accomplished at room temperature though the reaction proceeds much more slowly. A staining time of 6 to 8 h at 400 C gives a more satisfactory result than 24 h at room temperature. Overnight or longer staining at 40° C is not recommended; oxide formation is increased but there is no marked increase in the deposition of metallic silver at the specific sites. Also, after about 18 h at 400 C a second side-reaction begins to be evident. If slides bearing squashes (or clean slides) are incubated in the reagent for 18 to 24 h or longer, there appears to be some reaction with the glass resulting in the deposition of a brownish film which cannot be removed by treatment with dilute ammonium hydroxide (metallic silver?). This reaction has not been observed with slides processed according to the recommended schedule. There is a considerable literature concerning the use of silver reagents in cytology and histochemistry. The studies most pertinent to those described here are those bearing on the specificity of ammoniacal silver solutions for aldehyde groups. Feulgen and Voit (1924) used ammoniacal solutions in their studies of the Schiff reaction. They demonstrated specificity by showing that the typical nucleal reaction was blocked by prior treatment of the hydrolysed tissue with an ammoniacal silver solution. Danielli (1949) made use of a similar reagent in his studies of aldehydes in liver tissue. The distribution of silver paralleled the distribution of aldehydes as visualized with other specific reagents. The PAS reaction affords indirect evidence of the specificity of ammoniacal silver solutions for aldehyde groups. Thus 1:2 glycol groups oxidized to aldehydes (by periodic acid, chromic acid, or other oxidizing agents) may be visualized by use of the Schiff reaction. Ammoniacal silver solutions have been substituted for the more usual Schiff reagent by Gomori (1946) and others. The results obtained paralleled those produced by the usual Schiff

FIG. 1 (plate), A to c, Melanoplus sp.; H, Chironomus sp.; A and B, Feulgen stain; c to H silver-Schiff. A, diplotene. B, metaphase. c, diplotene. D, diakinesis. E, metaphase. F, highly endoploid interphases from testicular sheath; note numerous masses of hetero- chromatin. G, portion of a bundle of needle-shaped sperm heads. Tails are barely visible at upper right. H, lightly squashed salivary gland nucleus; note well defined banding patterns. 366 Bryan—A silver-Schiff reaction reagent. The reagent described here gives essentially the same results as the foregoing when used in the PAS procedure. The usual squash procedures result in either the almost complete dis- ruption and loss of cytoplasm or, at least, a redistribution of it in the form of a thin film. For these reasons cytoplasmic staining, if present at all, is barely perceptible and of no material significance. However, when dealing with sectioned material residual cytoplasmic aldehydes, or the presence of other substances capable of reducing the silver complex are of more concern. Fortunately, it is known from studies of the Feulgen reaction that acid hydrolysis removes certain substances capable of reacting with aldehyde reagents, and that fixation in lipid solvents such as acetic acid and alcohol also removes the bulk of substances which give rise to the so-called plasmal reaction (e.g. acetal phosphatides). Acetic alcohol or similar fixatives are, therefore, the fixatives of choice for this procedure. Neutral formalin or dichromate- containing fixatives (which better preserve tissue lipids) contribute to the problem of cytoplasmic staining. Thus unhydrolysed sections of Helly- fixed mouse testis showed relatively pronounced cytoplasmic staining, with the acrosomes of developing spermatids showing a somewhat higher density of silver than the rest of the cytoplasm. The foregoing observations suggest that the silver-Schiff procedure, as described here, may be somewhat more sensitive than the usual Feulgen reagent to conditions favouring 'non-specific' staining. This conclusion does not, however, rule out its potential usefulness in chromosome studies.

This investigation was supported by Public Health Service Research Grant CA-05591-02 from the National Cancer Institute.

References Bradfield, J. R. G., 1954. Nature, Lond., 173, 134. Bretschneider, L. H., 1949. Proc. Acad. Sci. Amst., 52, 301. Conger, A. D., and Fairchild, L. M., 1953. Stain Tech., 28, 281. Danielli, J. F., 1949. Quart. J. micr. Sci., 90, 67. Feulgen, R., and Voit, K., 1924. Hoppe-Seyl. Z., 135, 249. Gomori, G., 1946. J. clin. Path., 16, 347. Jurand, A., Deutsch, K., and Dunn, A. E. G., 1959. J. R. micr. Soc, 78, 46. Mellor, J. W., 1939. Modern inorganic chemistry, revised and edited by G. D. Parkes, p. 601. London (Longmans, Green & Co.). Smith, G. S., 1943. J. Path. Bact., 55, 227.