445 The vital staining of Amoeba proteus By JENNIFER M. BYRNE (From the Cytological Laboratory, Department of Zoology, University Museum, Oxford) With one plate (fig. 2) Summary The effect of keeping Amoeba proteus in dilute basic dye solutions was studied. It was found that Nile blue, neutral red, and neutral violet in particular, and also brilliant cresyl blue, methylene blue, Bismarck brown, thionin, toluidine blue, and azures A and B act as vital dyes, while at comparable molarities crystal violet, dahlia, safranin, methyl green, Janus green, and Victoria blue are lethal, and do not produce any stain- ing until after death. Azure C, basic fuchsin, and particularly pyronine G are relatively harmless, but produce no vital staining. All the vital dyes stain the food vacuoles, and all produce small, darkly stained granules in colourless vacuoles in the cytoplasm. The latter do not exist in the unstained amoeba. Some of the dyes colour vacuoles around the crystals. These crystal vacuoles also seem to be induced. A few of the dyes colour the spherical refractive bodies, which are at least in part phospholipid. All the basic dyes used with the possible exception of azure C, methyl green, and pyronine G attach to the external membrane of A. proteus in an orientated manner, as shown by the increase in birefringence of the external membrane induced by thess dyes. It is particularly those dyes that act as vital dyes that produce a very pronounced increase in the birefringence of the external membrane. Introduction MOST dyes which can be used to colour pre-existing cell inclusions in life are basic dyes, as pointed out by Fischel (1901) and von Mollendorff (1918). But not all basic dyes can be used as vital dyes, nor do the known vital dyes belong to any particular chemical group. A number of generalizations about the chemical composition and properties of vital dyes have been made (Overton, 1890, 1900; Fischel, 1901; Heidenhain, 1907; Irwin, 1928; Brooks and Brooks, 1932; Seki, 1933), but in fact it does not seem to be possible to generalize in simple terms. The ability of a dye to penetrate a cell, its toxicity, and its ability to stain specific inclusions within the cell must be considered separately. A series of experiments was performed on Amoeba proteus Leidy with a number of basic dyes, both vital and non-vital, to find out if the non-vital dyes failed to produce a vital colouring because they were lethal to the organ- ism or because, while harmless, they either did not penetrate at all, or did not penetrate in quantities sufficient to produce any visible colouring. Mitchison (1950) showed that if living amoebae are placed in dilute solu- tions of certain basic dyes the natural birefringence of the external membrane is enhanced. This indicates that the dyes in question are orientated at the [Quart. J. micr. Sci., Vol. 104, pt. 4, pp. 445-58, 1963.] 2421.4 G g 446 Byrne—Vital staining of Amoeba proteus surface in an orderly molecular array. Observations were therefore made with the polarizing microscope to see if there was any correlation between those dyes which produced a vital colouring and those which were capable of attaching themselves in an orientated manner to the external membrane of the amoeba. Material and Methods The amoebae used in this work were of a strain of A. proteus maintained in wheat grain cultures in this Department for a number of years by Mr. P. L. Small. A number of basic dyes, both vital and non-vital, were tried (see appendix). The dyes were used in aqueous solution at concentrations of 3 X io~6 M, 1 x 10-5 M, 3 X 10-5 M, 1 X 10-4 M, 5 X io~4 M, and 1 X io~3 M. Two milli- litres of each dye solution were pipetted into a solid watch glass and 30 amoe- bae added with as little water as possible. This was achieved by sucking the amoebae into a pipette, which was then held vertically until all the amoebae had sunk to the tip and could be transferred in a single drop of water. The amoebae were examined 24 h after placing in the dye solution and subse- quently at 24-h intervals for periods up to 31 days. The amoebae were placed on a slide with a coverslip supported by two other coverslips and examined microscopically under the oil-immersion objective. Observations were made on the cytoplasmic inclusions of A. proteus by means of the Baker interference microscope. In order to prevent any pressure on the amoebae, each was placed in a drop of water in a cavity slide and a coverslip applied. The whole was then quickly inverted so that the amoebae fell on to the coverslip. The amoebae were left to attach to, and begin moving on the coverslip, at which stage the slide could be inverted again and the amoebae studied on the coverslip without any danger of applying pressure to them. The Baker double-focus water-immersion objective, NA 1-3, was used. The acid haematein (AH) test for phospholipids (Baker, 1946, 1947) and the periodic acid / Schiff (PAS) test for carbohydrates (McManus, 1948) were performed on fixed amoebae. For the AH test the amoebae were fixed, postchromed, and embedded in gelatine in small glass tubes, the amoebae being centrifuged down between each operation. After the gelatine had solidified the tube was broken away. Ten-micron sections were cut on the freezing microtome. For the PAS test the amoebae were suspended in a concentrated solution of bovine plasma albumin and embedded in a piece of junket according to the method developed by Ross (1961) for ascites tumour cells; they were then fixed in formaldehyde-calcium (Baker, 1944). Polarized light observations were made to determine which of the dyes used attached themselves in an orderly fashion to the external membrane of A. proteus. Both acid and basic dyes were used in aqueous solutions of 1 X 10-4 M, 5 x 10-4 M, 1X 10-3 M, and 5 X io"3 M (see table 6). The amoebae Byrne—Vital staining of Amoeba proteus 447 were left in the dye solutions for 5 to 30 min and then examined under a Swift polarizing microscope, with a 4-mm objective. Results Microscopical examination, including interference microscopy, shows the cytoplasmic inclusions of A. proteus to comprise food vacuoles of various sizes, containing food in various stages of digestion, a large number of bipyramidal crystals varying from 2 to 7 /x in length, and a large number of a~granules crystal spherical refractive •acuole small granules food vacuole mitochondrion crystal vacuole FIG. 1. A, diagram of the cytoplasmic inclusions of A. proteus. B, diagram of the cytoplasm of A. proteus after staining with a vital dye. spherical refractive bodies up to 7 ju, in diameter. Mast (1926) described 'refractive spherical bodies' in A. proteus. Andresen (1942) found similar structures in the cytoplasm of Chaos chaos and renamed them 'heavy spherical bodies'. Pappas (1954) uses the term 'spherical refractive bodies'. There are two other types of inclusion, the mitochondria and the 'a-granules' of Mast (1926). The mitochondria ('^-granules' of Mast) are more or less spherical and about 1 /A in diameter. The a-granules are about 0-25 /x in diameter, and are of unknown composition. A. proteus has a single large contractile vacuole, surrounded by a layer of mitochondria. A diagrammatic representation of the cytoplasmic inclusions of A. proteus can be seen in fig. 1, A. Interference microscope observations Carefully handled A. proteus observed by means of the interference micro- scope in general do not show vacuoles around the crystals (figs. 1, A; 2, A). But vacuoles appear very quickly, often within 3 to 5 min, in the beam of the microscope lamp (fig. 2, B, c). When vacuoles are present they can be seen very easily with the interference system because they are of lower refractive index than the ground cytoplasm. If a heat-absorbing filter (Chance ON 22) 448 Byrne—Vital staining of Amoeba proteus is used, the amoebae can be observed for an hour without crystal vacuoles appearing. This indicates that the heat rather than the light from the lamp is responsible for the induction of the vacuoles. Pressure also seems to induce the formation of vacuoles. Occasionally an amoeba mounted under an un- supported coverslip does not show crystal vacuoles. If gentle pressure is applied by racking the objective down a little, large vacuoles immediately appear. (Dyes also cause the appearance of vacuoles. See below.) Histochemistry The spherical refractive bodies are coloured blue by the AH test (Baker, 1946). After pyridine extraction (Baker, 1947) they are colourless. These findings indicate the presence of phospholipid. No other inclusion gives a positive reaction to the AH test. The spherical refractive bodies are nega- tive to the PAS test (McManus, 1948). Vital staining The results of keeping A. proteus in dilute basic dye solutions can be seen in tables 1 to 5 (see appendix). At the lowest concentration of dye used (3 X io~6 M—see tables 1 and 2), only Nile blue, neutral red, and neutral violet act as vital dyes. All three stain the food vacuoles within 24 h. The contents of the food vacuoles stain slightly darker than the vacuolar fluid. Neutral red colours vacuoles around the crystals (see fig. 1, B) orange-red after 4 days; neutral violet colours them after 13 days. The amoebae remain active in the neutral red and neutral violet solutions for 28 days or more. Nile blue at the same molarity stains the spherical refractive bodies dark blue in 24 h and the crystal vacuoles pale blue in 2 days.