421 A New Method for Staining Connective Tissue Fibres, with a Note on Liang's Method for Nerve-fibres By G. OWEN (From the Department of Zoology, The University, Glasgow) With two plates (figs, i and 2) SUMMARY A new method of staining connective tissue fibres is described. The stain used is alcohol-soluble aniline blue (spirit blue), after oxidation with potassium permanganate. The method is rapid and easy in application. Liang's method for staining nerve-fibres is discussed briefly and its use in demonstrating the excretory system of Fasciola described. INTRODUCTION /CONNECTIVE tissue fibres, particularly the finer reticular fibres, are V_>< often demonstrated by silver impregnation techniques. In the majority of these methods the sections are first oxidized with potassium permanganate and decolorized by a rinse in sulphurous acid before metallic impregnation. In the method outlined here, sections are similarly treated with permanganate and sulphurous acid, but this is followed by a brief period in alcohol-soluble aniline blue, a stain rarely used in histological techniques. While not the equal of successful silver preparations, the method has proved simple and rapid in use, and demonstrates the distribution of connective tissue fibres in a variety of animals. Fig. 1 illustrates the sub-epidermal region of A, a nemertine; B, Lumbricus; c, D, and E, Peripatus; and F, an ammocoete larva. The distribution of fibres in the sub-epidermal region of a nemertine (Amphiporus) has been described in detail by Cowey (1952) and it would appear that a somewhat similar system of fibres occurs in the earthworm. After treatment with the aniline-blue method outlined here, a system of fine reticular fibres can be demonstrated, particularly in the region of the circular muscle-layer (fig. 1, B). Sections of Peripatus and of ammocoete larvae treated in this way proved most interesting. Fig. 1, c shows a section of Peripatus treated with Mallory's triple stain. Beneath the epidermis of the integument is a layer of connective tissue, 15 /J, thick, which colours blue and has a more or less homogenous appearance. After treatment with permanganate and spirit blue, the bulk of this layer remains colourless, or is coloured green if light green is used as a counterstain; but traversing the layer can now be seen numerous 'struts' of fine fibres coloured a deep blue (fig. 1, D). Beneath the epidermis these fibres are continuous with those of the basement membrane, while internally they are continuous with a system of fine anastomosing fibres surrounding the individual [Quarterly Journal of Microscopical Science, Vol. 100, part 3, pp. 421-424, Sept. 1959.] 421.3 F f
422 Owen Method for Staining Connective Tissue Fibres muscle-fibres (fig. i, E). This connective tissue 'skeleton' of Peripatus is of considerable interest in the light of Manton's (1958) discussion of habits and evolution of body design in the arthropods. A somewhat similar system of fibres can be demonstrated in the sub-epidermal connective tissue of ammocoete larvae (fig. 1, F). Figs. 2, A, B, C, illustrate preparations of various vertebrate tissues. Fig. 2, A shows the fibres in the submucosa of the frog's stomach; B, those of the muscularis externa of the small intestine of the cat; and C, those of the diaphragm of the cat. The advantages of the permanganate / spirit blue method are its simplicity and its application to tissues fixed in a variety of fixatives. It is interesting to note that the method also stains neurosecretory material with results somewhat similar to those obtained with the paraldehyde/fuchsin technique (Gabe, 1953). This stain was used earlier by Gomori (1950) for elastic fibres, which are also demonstrated by the present method (fig. 2, A). o.. METHOD Solutions Oxidizing solution Potassium permanganate. 0-5 g Cone, sulphuric acid.. 0-5 ml Water..... 100 ml This solution remains effective for approximately 2 h. The concentration of potassium permanganate is not critical. Decolorizing solution Potassium bisulphite Normal hydrochloric acid Water.... Stain solution Alcohol-soluble aniline blue ('michrome' brand) Aniline oil. 70% alcohol. i-og i-o ml. 99 ml. 0-5-1-0 g i-o ml. 99 ml The concentration of the stain can be varied to suit various tissues. FIG. 1 (plate). A, sub-epidermal region of a hoplonemertine. Fixed in Bouin's fluid and treated with potassium permanganate spirit blue. B, T.S. Lumbricus, showing connective tissue fibres traversing the layer of circular muscle. Fixed and stained as in A. c, T.S. Peripatus, showing the integument, connective tissue layer, and circular muscle. Fixed in modified Bouin-Dubosq (Atkins, 1937) followed by Mallory's triple stain. D, as c but treated with potassium permanganate / spirit blue in place of Mallory's triple stain. Note the 'strut'-like system of fibres traversing the layer of connective tissue. E, staining as D but showing the connexion between the 'strut'-like fibres and the system of fibres investing a circular muscle fibre. F, sub-epidermal region of an ammocoete larva. Fixed in Bouin's fluid and stained as in D.
A t B. 20 M, C 6 D E t F Fie. i G. OWEN
* FIG. 2 G. OWEN
Owen Method for Staining Connective Tissue Fibres 423 Procedure 1. Bring sections to water in the usual way. 2. Oxidize in the potassium permanganate solution for 1 min. 3. Rinse in distilled water (30 sec). 4. Decolorize in sulphite solution (approximately 30 sec). 5. Rinse well in distilled water (1-2 min). 6. Stain in aniline blue (2 min). 7. Rinse in water to remove excess stain. 8. Transfer directly to absolute alcohol and differentiate (with some tissues this may take place rapidly). 9. Clear in xylene and mount in balsam. The tissues will have a general background colour of light blue while connective tissue fibres will be coloured intense blue. A solution of light green SS (0-5-1-0% in 1% aqueous acetic acid) may be used as a counterstain (approximately 5 sec) after stage 7. If this is used, the time of differentiation in absolute alcohol is reduced. Liang's method of using the Schijf reaction for nerve staining Liang (1947) reported a new method for staining nerve-fibres and endings with the Schiff reagent. This has been used in this Department and the following comments are of interest. It should be pointed out that the method has not proved successful in demonstrating motor-nerve end-plates. Nevertheless, it has provided a simple and rapid method of demonstrating the general distribution of nerve-fibres in a variety of animals and tissues. In particular, it has been used successfully with bivalve and gastropod molluscs, animals in which it is notoriously difficult to demonstrate the peripheral nervous system with the more familiar methylene blue and metallic impregnation techniques. Fig. 2, E is a portion of a whole mount of the siphon of Buccinum (Gastropoda) prepared by Mr. P. S. Maitland, of this Department, by using the Schiff reagent. It demonstrates most clearly the rich innervation of this organ. For the preparation of whole mounts, or imbedding in wax and subsequent sectioning, Liang recommended rapid dehydration in the alcohols. It has been found, however, that the purple colour of the fine nerve-fibres is rapidly removed in both alcohol and cellosolve, and it is not possible to obtain successful preparations in this way. Instead, tissues should be transferred directly to several changes of absolute acetone and then to xylene. Mount in balsam FIG. 2 (plate). A, submucosa of frog's stomach. Fixed in i % formic acid and treated with potassium permanganate / spirit blue. B, muscularis externa of the small intestine of the cat. Fixed in Zenker's fluid; staining as in A. c, diaphragm of cat. Fixed in i % formic acid; staining as in A. D, excretory system of Fasciola hepatica (whole mount). Fixed in i% formic acid; staining by Liang's method but dehydrated in absolute acetone; cleared in xylene and mounted in balsam. E, siphon of Buccinum undatum showing nerve supply (whole mount). Treatment as in D.
424 Owen Method for Staining Connective Tissue Fibres or embed in wax. The whole mount of the siphon of Buccinum (fig. 2, E) was prepared in this way. One further point is of interest. Fresh specimens of the liver fluke, Fasciola, were subjected to Liang's method but failed to show any trace of the nervous system. It was successful, however, in demonstrating in detail the excretory system, a portion of which is shown in fig. 2, D. The granular contents of the peripheral excretory ducts are coloured an intense red. REFERENCES ATKINS, D., 1937. 'On the ciliary mechanisms and inter-relationships of lamellibranchs. Pt. IV. Cuticular fusion with special reference to the fourth pallial Aperture in certain lamellibranch.' Quart. J. micr. Sci., 79, 423. COWEY, J. B., 1952. 'The structure and function of the basement membrane muscle system in Amphiporus lactifloreus (Nemertea)'. Ibid, 93, 1. GABE, M., 1953. 'Sur quelques applications de la coloration par la fuchsine-paraldehyde.' Bull. Micr. appl., 3, 154. GOMORI, G., 1950. 'Aldehyde fuchsin; a new stain for elastic tissue.' Amer. J. clin. Path., 20, 665. LIANG, H. M., 1947. 'A new method for staining nerve fibres and endings using the Schiff reaction.' Anat. Rec, 97, 419. MANTON, S. M., 1958. 'Habits of life and evolution of body design in Arthropoda.' J. Linn. Soc. (Zool.), 44, No. 295, 58.