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. Amoebae stained with Nile blue show within 24 h a number of dark blue granules about 0-5 to 0-75 /x in diameter in colourless vacuoles 2-5 to 3-o /x in diameter (see fig. 1, B). The granules are single at first, but with increased staining the number of granules in each vacuole, and the total number of vacuoles increases. The amoebae remain active in Nile blue solutions for 10 days. At 3 X io~6 M, Bismarck brown, brilliant cresyl blue, methylene blue, and thionin produce a very faint staining of the food vacuoles in some, but not in all specimens within 1 to 2 days. No other inclusions are stained. The amoebae remain active in these dyes for 21 days or more. Crystal violet, dahlia, and safranin are lethal within 3 to 4 days at this molarity, as are to a slightly lesser extent (8 to 12 days) methyl green, Janus green, and Victoria blue. None of these dyes acts as a vital dye on amoebae.

FIG. 2 (plate). Interference microscope photographs of A. proteus. A, crystals lying free in the cytoplasm. B and c, crystals surrounded by crystal vacuoles. cr, crystal; crv, crystal vacuole. FIG. a J. M. BYRNE Byrne—Vital staining of Amoeba protens 449 At the same molarity, toluidine blue, the azures, basic fuchsin, and pyro- nine G do not stain any of the inclusions of the amoebae. The amoebae remain active in these dyes for periods of 17 days or more. At a slightly increased molarity (1X io~s M—see tables 1 and 3) Nile blue, neutral red, and neutral violet are the most effective vital dyes as before, but Nile blue is rather toxic. The amoebae are sluggish after 24 h in this dye solution, and begin to round off after a few days. Nile blue stains the food vacuoles, the crystal vacuoles, and the spherical refractive bodies within 24 b. The amoebae also show within 24 h numerous small darkly stained granules 0-5 to i-OjU, in diameter in colourless vacuoles 3-5 104-5 fj, in diameter. Neutral red and neutral violet stain the food vacuoles within 24 h as before. Both dyes colour the crystal vacuoles in 3 days, and stain some of the spherical refrac- tive bodies dark red after 13 days. Amoebae kept in these dyes show numerous small dark red granules in colourless vacuoles in the cytoplasm within 24 h in neutral red, and within 2 days in neutral violet. The granules are similar to those found with Nile blue, and at first measure 0-5 to i-o JX in diameter in vacuoles 3-5 to 4-5 /x in diameter. There are usually 2 or 3 granules in each vacuole. With increased lengths of time in the dye solutions the size of the granules increases to 1*5 /A, and the number of granules in each vacuole increases to 5 or 6. The amoebae remain active in these dyes for 20 days or more. Methylene blue, Bismarck brown, brilliant cresyl blue, and after 4 days, thionin, prove to be vital dyes at this concentration. All stain the food vacuoles. Brilliant cresyl blue stains the crystal vacuoles pale blue in 8 days. Pale blue crystal vacuoles were found in one specimen stained with methylene blue, but this seems to have been exceptional. Amoebae stained with methy- lene blue show after 24 h a few small dark blue granules, similar to those found with Nile blue or neutral red, 0-5 to 0-75 fj, in diameter, in colourless vacuoles 2-0 to 3-0 /x in diameter. The amoebae remain active in these dye solutions for 10 to 14 days. Toluidine blue and azure A at the same molarity stain the food vacuoles in some, but not in all specimens. The amoebae remain active in these solutions for 16 days or more. Azures B and C, basic fuchsin, and pyronine G at the same molarity do not act as vital dyes and are non-toxic. The amoebae remain active for 17 days or more (30 days in the case of pyronine G). With further increase in molarity (3 X io~5 M—see tables 1 and 4) Nile blue becomes very toxic. The amoebae are rounded off after 24 h and are killed within the next 24 h. The staining is the same as with the lower concentrations of dye, except that the external surface of the amoeba is distinctly stained blue. Neutral red and neutral violet are also toxic at this concentration. Neutral red kills the organisms within 3 to 4 days, and neutral violet within 8 days. Neutral red stains the food vacuoles, the crystal vacuoles, and the spherical refractive bodies in 24 h. The amoebae also show within 24 h large numbers of small, dark red granules. The granules measure 075 to 1-5 JU. in 45° Byrne—Vital staining of Amoeba proteus diameter and are found in clusters of io or 12 granules in colourless vacuoles 3 -o to 5 -o /n in diameter. Neutral violet stains the food vacuoles and the crystal vacuoles in 24 h. Some of the spherical refractive bodies stain faintly in 2 days, and all are deeply stained after 5 to 6 days. The amoebae show many small stained granules after 2 days, exactly similar to those found after the use of neutral red. At 3 X io~5 M, methylene blue, brilliant cresyl blue, Bismarck brown, thionin, toluidine blue, and azure A stain the food vacuoles within 24 h. Brilliant cresyl blue stains the crystal vacuoles pale blue in 24 h. Amoebae stained with methylene blue, brilliant cresyl blue, and toluidine blue show in 24 h large numbers of small darkly stained granules in clusters of up to 15 granules in colourless vacuoles in the cytoplasm. Those stained with thionin and azure A show a few darkly stained granules in colourless vacuoles, the granules usually single or paired. All five dyes are lethal at this concentration. Thionin and toluidine blue kill the amoebae in 3 days; methylene blue and brilliant cresyl blue in 4 days, and azure A in 5 days. Methylene blue, toluidine blue and azure A stain the external surface of the amoebae. Toluidine blue and azure A stain metachromatically. Some amoebae kept in Bismarck brown show a few, small, very pale brown granules in colourless vacuoles after 4 days. The granules measure 0-5 to i-o JU. in diameter and are usually single. The amoebae remain alive for 15 days or more in this dye. Azure B at the same molarity stains occasional food vacuoles in some speci- mens, but in general does not act as a vital dye. The amoebae remain active for 14 days or more. Azure C, basic fuchsin, and pyronine G do not produce a vital colouring. Basic fuchsin is rather toxic at this concentration. The amoebae die after 6 to 7 days. But azure C and pyronine G seem harmless. The amoebae remain active in azure C solutions for 14 days or more, and in pyronine G solutions for up to 30 days. Azures A, B, and C, Bismarck brown, basic fuchsin, and pyronine G were tried at 1 X io~4 M (see tables 1 and 5). Bismarck brown and azure A stain the food vacuoles in 24 h as before. Azure A also stains the crystal vacuoles in 3 days. At this concentration the amoebae also show large numbers of small, deep purple granules in colourless vacuoles. The edge of the amoeba stains pinkish. The dye is toxic at this molarity and the animals are killed in 4 days. Amoebae kept in Bismarck brown show some colourless vacuoles containing single deeply stained granules. They do not occur in all specimens. The external membrane stains brown at this concentration. The amoebae die in 7 to 8 days. Azure B definitely acts as a vital dye at 1 X io~4 M. The dye stains some of the food vacuoles in 24 h, and stains them all pale purplish blue in 2 days. The amoebae also show a few deep purple granules in colourless vacuoles after 24 h. The granules are mostly single or paired. The amoebae remain active in this dye for 10 days or more. Basic fuchsin is toxic at this molarity. The amoebae die in 3 to 4 days. There is no vital staining. Byrne—Vital staining of Amoeba proteus 451 Pyronine G and azure C do not stain the amoebae. They are not toxic. The amoebae remain active for 15 to 20 days or more. Azures B and C, Bismarck brown, and pyronine G were tried at further increased molarity (5 X io~4 M—see tables 1 and 5). Bismarck brown is toxic. The animals are killed within 24 h. Azure B stains the food vacuoles purplish blue within 24 h, and produces a large number of darkly stained granules in colourless vacuoles. Each vacuole contains 4 to 6 granules. The amoebae round off after 4 days. Azure C and pyronine G produce no staining at this concentration. The amoebae remain active for 10 days in azure C solutions and for 20 days or more in pyronine G. Increasing the molarity of azure B to 1 X io~3 M produces staining of the food vacuoles within 24 h, as at lower concentrations. A large number of deeply stained granules in colourless vacuoles is also produced, up to 15 granules in each vacuole. The edge of the amoeba is stained pinkish, and the cytoplasm appears pinkish, although the crystal vacuoles do not seem to stain. The amoebae are killed in 2 to 3 days. The amoebae are killed in azure C solutions at this molarity after 4 to 6 days. There is no staining until after death. Increasing the molarity of pyronine G to 1 X io~3 M still has no effect, the amoebae remain active for 30 days. After 14 days in this concentration of dye none of the inclusions are stained and the amoebae do not show any darkly stained granules in vacuoles, but some specimens have clear, pink vacuoles 15 to 25 /x in diameter, often occurring near the contractile vacuole. Further increase in molarity to 5 X io~3 M induces pinocytosis (see table 6) and the amoeba dies in a few hours. None of the dyes used stains either the mitochondria or the a-granules. Polarized light observations The results of the observations with the polarizing microscope can be seen in table 6. All the basic dyes tried, with the possible exception of azure C, methyl green, and pyronine G, produce an increase in the birefringence of the external membrane of living A. proteus when viewed between crossed polaroids, although the degree to which the effect is developed varies greatly. The colour as seen in the non-compensated microscope is greenish yellow. The effect disappears on death. At the concentrations used in these experiments the dyes stain the external membrane of the amoeba as seen with the ordinary light microscope. It should be noted that the metachromatic dyes stain with their metachromatic colour. Methyl green and pyronine G stain the external membrane but only possibly produce a very slight increase in birefringence. Azure C, even used in saturated solution, does not produce a visible staining of the membrane. After 30 min staining with the saturated solution it possibly produces a very slight increase in birefringence. None of the acid dyes tried, including the anomalously acting eosin group, either stained the external membrane in life, or produced an increased 452 Byrne—Vital staining of Amoeba proteus birefringence. Aurantia produced an increased birefringence of the whole animal coincident with total staining on death. At the concentrations used in these experiments, the basic dyes with the exception of azure C induced pinocytosis, but it was observed only very occasionally with basic fuchsin, Bismarck brown, dahlia, and Victoria blue. Discussion The results of keeping A. proteus in various dilute basic dye solutions sharply mark off neutral red, neutral violet, and Nile blue in particular, and also methylene blue, brilliant cresyl blue, Bismarck brown, thionin, toluidine blue, and azures A and B from the other basic dyes tried. All these dyes act as vital dyes on A. proteus, although the number of inclusions that each dye will stain, and the molarity at which each dye will stain a given inclusion vary widely. Crystal violet, safranin, dahlia, methyl green, Janus green, and Victoria blue are very lethal at comparable molarities, and produce no staining until after death. Andresen (1942) found dilute solutions of Janus green to be lethal to C. chaos. Duijn (1961) has shown that bull spermatozoa stained with Janus green and exposed to light show decreased movement. Basic fuchsin, azure C, and particularly pyronine G are relatively non-toxic, but produce no staining. All the dyes found to act as vital dyes first stain the food vacuoles. All stain the contents darker than the vacuolar fluid. All the vital dyes also pro- duce small deeply stained granules in colourless vacuoles in the cytoplasm. These granules have been observed in A. proteus after the use of neutral red by Andresen (1946) and Pappas (1954). Andresen (1942, 1945) and Torch (1959) found similar granules in Pelomyxa carolinensis (C. chaos) after staining with neutral red. Andresen (1942) also reported similar granules in C. chaos after the use of Nile blue, brilliant cresyl blue, and toluidine blue. With all the vital dyes except Bismarck brown the number of vacuoles, number of granules per vacuole, and the size of the granules and the vacuoles increases with increased length of time of staining, and with increase in the concentration of the dye. This has also been observed by Andresen (1942, 1945, 1946), Pappas (1954), and Torch (1959). After the use of Bismarck brown the granules are very few, and occur singly or paired in each vacuole even at lethal concentrations of dye. Some specimens show no granules. Andresen (1942) also found that Bismarck brown did not produce granules in all speci- mens. The interference microscope shows nothing in the unstained animal corresponding to these granules in vacuoles in the cytoplasm. The only inclusions of comparable size are the a-granules and the mitochondria. These remain unstained during vital dyeing, and also are never found in vacuoles. These facts and the increase in size and number of the granules during staining strongly suggests that the granules arise under the influence of the dye. This conclusion has also been reached by Andresen (1942, 1945, 1946), Pappas (1954), and Torch (1959). Goldacre (1952) considers such granules Byrne—Vital staining of Amoeba proteus 453 to be a precipitation effect in the cytoplasm. Perhaps, as suggested by Torch, the formation of these granules^ represents a protective mechanism against the toxicity of the dye, precipitation removing the dye from the cytoplasm. If granule-formation is a protective mechanism, the absence of these granules in amoebae kept in the non-vital dyes (either lethal like crystal violet or relatively harmless at comparable molarities like pyronine G) may be evidence that neither of these groups of dyes penetrates the amoebae at all in life. This would mean that the lethal dyes must be entirely surface-acting. Staining of the crystal vacuoles of A. proteus was observed by Vonwiller (1913), Edwards (1924), Mast (1926), Koehring (1930), Mast and Doyle (1935), Andresen (1946), Pappas (1954), and Noland (1957), and of Pelomyxa by Andresen (1942,1945), Wilber (1942), and Torch (1959). Andresen (1942), and Wilber (1942) find that Nile blue stains the crystal vacuoles in C. chaos. Vonwiller (1913) reported the staining of the crystal vacuoles of A. proteus with methylene blue, but I have observed this only exceptionally (see table 3). Hofer (1890), and Schubotz (1905) find that the crystal vacuoles of A. proteus stain with Bismarck brown, but I have not seen this. Andresen (1942) stained the crystal vacuoles of C. chaos with Bismarck brown. Singh (1938) did not find crystal vacuoles in his strain of A. proteus, and Allen (1961) believes that the crystals of A. proteus, like those of A. dubia, lie free in the cytoplasm in carefully handled, uncompressed amoebae. In A. dubia vacuoles can be induced to form around the crystals by compression under a coverslip, exposure to heat and intense light, and by fixation and centrifugation. My observations on A. proteus with the interference micro- scope support this view. In carefully handled, uncompressed amoebae there are no crystal vacuoles, but they are rapidly induced by the heat of the micro- scope lamp, or by pressure on the coverslip. Vital dyes must be added to the list of agents inducing the formation of crystal vacuoles. The crystals have recently been shown (Griffin, i960; Carlstrom and Moller, 1961) to be an excretory product, carbonyl diurea (triuret). Allen (1961) suggests that the crystal forms a focus for vacuolar formation. Perhaps since the crystals themselves are an excretion, the appearance of stained, vacuoles around them marks sites of elimination of the dye from the cyto- plasm. It would be interesting to know whether the dyes which do not act as vital dyes also produce crystal vacuoles even if they are not visibly stained, because this would reveal whether or not these dyes penetrate the amoeba at all in life, or whether, as suggested before, the lethal dyes are surface-acting. However, because of the ease with which crystal vacuoles can be induced, it is impossible to get a definite answer to this point. Of the vital dyes, only Nile blue, neutral red, and neutral violet stain the spherical refractive bodies. Staining of these inclusions in A. proteus with neutral red has been noted by Mast (1926), Mast and Doyle (1932, 1935), Singh (1938), Andresen (1942), and by Pappas (1954). Andresen (1946), however, found that they stained only exceptionally in living A. proteus. Vonwiller (1913) found that the 'Eiweisskiigeln' of A. proteus stained vitally 454 Byrne—Vital staining of Amoeba proteus with neutral red and Bismarck brown. These inclusions seem to be identical with the spherical refractive bodies, although I do not find that they stain with Bismarck brown. The spherical refractive bodies are at least in part phospholipid. Mast and Doyle (1935) found protein and lipid in the outer layer of the spherical refractive body. This was confirmed by Pappas (1954). Heller and Kopac (1955) determined the presence of an organic phosphate component in the cortex of the spherical refractive body, and the positive reaction to the AH test is in accord with this. Mast and Doyle believed that the inner shell of the spherical refractive body contained carbohydrate. However, Pappas (1954) found no reaction either with the PAS test or with Lugol's solution for starch. I also find the spherical refractive bodies PAS-negative. It has been mentioned in a previous paper (Byrne, 1962) that there is a tendency for pre-existing cellular inclusions that colour with vital dyes to be wholly or partly phospholipid. The staining of the spherical refractive bodies is another instance of this. It is not evident why only Nile blue, neutral red, and neutral violet, and not the other vital dyes, stain the spherical refractive bodies. Only the staining of the food vacuoles and the spherical refractive bodies is a true vital staining. The small granules in vacuoles are an artifact of the dye, as is the induction of the crystal vacuoles. The induction of pinocytosis in A. proteus with toluidine blue and brilliant cresyl blue has also been noted by Quertier and Brachet (1959), and with toluidine blue by Rustad (1959, 1961). The metachromatic staining of the external membrane of amoeba by basic dyes at the concentrations used in the polarized light experiments has been noted by Spek and Gillissen (1943) and Rustad (1961). Partly because of this metachromasia the site of attach- ment of the dyes and other pinocytotic inducers is thought to be an acidic mucopolysaccharide layer (Lehmann, Manni, and Bairati, 1956; Marshall, Schumaker, and Brandt, 1959; Bell, 1961; Nachmias and Marshall, 1961; Rustad, 1961). Goldacre and Lorch (1950), Prescott (1953), and Noland (1957) find that in o-oi to o-ooi% solutions of neutral red and methylene blue it is always the rear of an activity streaming amoeba that accumulates dye, while motionless amoebae stain uniformly around the periphery. Goldacre and Lorch (1950) and Goldacre (1952, 1961) relate this to their theory of amoeboid movement according to which the cortical gel component of the cytoplasm converts to the sol condition at the rear of the animal. According to this theory the dye is taken up on unsatisfied bonds of protein molecules in the cortical gel and plasma membrane, the dye being shed into the interior of the amoeba when the molecules fold into the sol configuration. The same mechanism for dye accumulation would operate in lower concentrations of dye solution. Wolpert and O'Neill (1962) find that there is no rapid turnover of surface membrane in A. proteus, and the differential staining found by Goldacre and others may be a function, not of accumulation by proteins during streaming, but of Byrne—Vital staining of Amoeba proteus 455 a membrane potential gradient along the organism (Bingley and Thompson, 1962; Bingley, Bell, and Jeon, 1962). Wolpert and O'Neill (1962) find a slow turnover of labelled surface membrane in A. proteus which might suggest a method of entry of vital dyes. But they attribute this turnover to pinocytosis at the tail, and vital staining takes place at much lower concentrations of dye than will induce pinocytosis. The polarization studies show that there is not an absolute correlation between those dyes which attach themselves in an orientated manner to the external membrane of A. proteus, and those that are capable of producing a vital colouring. It is, however, striking that it is those dyes which act as vital dyes that produce a very pronounced increase in the birefringence of the membrane, and which must therefore be attached to the membrane in a highly organized manner. It would then seem that such attachment is a necessary pre-requisite of vital dyeing in amoeba.

I am indebted to Dr. J. R. Baker, F.R.S., and to Dr. S. Bradbury for valuable help and advice given during the course of this work, and to Professor J. W. S. Pringle, F.R.S., for accommodating me in his Department. I am most grateful to Mr. P. L. Small for providing me with cultures of A. proteus. This work was carried out during the tenure of a Medical Research Council Scholarship.

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Appendix TABLE I The action of basic dye solutions at various molarities on A. proteus

Concentration Dye 3 X io~G M i X io~5 M 3 X io~5 M i x io~4M S x io-" M i x io~sM Nile blue . + + t + 1 Neutral red. + + 1 Neutral violet + + 1 Brilliant cresy 1 blue ± + 1 Methylene blue . ± + + 1 Thionin ± -j- + 1 Bismarck brown . ± -|_ _|_ -)- Toluidine blue o ± + 1 Azure A o ± + t + 1 Azure B o 0 ± + + + 1 Azure C 0 0 o o 0 ol Pyronine G. o o o o o o Basic fuchsin o 0 ot ol Janus green ot ol Victoria blue ot ol Methyl green ot ol Dahlia ol Safranin ol Crystal violet ol Key: + = dye acts as vital dye; rb = dye acts as vital dye in some, but not all specimens; o = dye does not act as vital dye; t = dye very toxic; 1 = dye lethal. Byrne—Vital staining of Amoeba proteus 457 TABLE 2 The staining of cytoplasmic inclusions of A. proteus by basic dye solutions at 3 X io~6 M

Spherical Food vacuoles refractive Crystal Small Dye Contents Fluid bodies vacuoles gramdes Nile blue + + + + + + Neutral red . 0 0 Neutral violet + + + + + 0 0 Brilliant cresyl blue ± ± 0 0 Methylene blue . ± ± 0 0 Thionin ± ± 0 0 0 0 0 + + +

Bismarck brown . ± ± 0 0 0

TABLE 3 The staining of cytoplasmic inclusions of A. proteus by basic dye solutions at 1 X 10-5 M

Spherical Food vacuoles Crystal Small Dye Contents Fluid bodies vacuoles granules Nile blue . + + + + + + + 4.4.4. 4.4. + + + + Neutral red . + + + + 4-4-4. 4-4.4- 4-4- 4-4-4.4- Neutral violet + + + + 4-4-4- 4-4-4- 4- 4-4-4-4- Brilliant cresyl blue + + + 4-4- 0 4- 0 Methylene blue . + + + 4-4- 0 ± + + + + Thionin 4-4- 4- 0 0 0 Bismarck brown . 4-4- 4- 0 0 0 Toluidine blue ± + 0 0 0 Azure A ± ± 0 0 0

TABLE 4 The staining of cytoplasmic inclusions of A. proteus by basic dye solutions at 3 X 10-5 M

Spherical Food vacuoles refractive Crystal Stnall Dye Contents Fluid bodies vacuoles gramdes Nile blue . Neutral red . Neutral violet Brilliant cresyl blue Methylene blue . Thionin Bismarck brown . Toluidine blue Azure A Azure B Key: + + + + = intensely stained; + + + = strongly stained; + + = slightly stained; + = very slightly stained; ± = stained in some, but not in all specimens; o = not stained. 8 Byrne—Vital staining of Amoeba proteus TABLE 5 The staining of cytoplasmic inclusions of A. proteus by basic dye solutions at 1X 10-4 M, 5 X 10-4 M, and 1 X 10-3 M Spherical Food vacuoles Small Dye Contents Fluid bodies vacuoles granules Bismarck brown + + + + + 0 0 ±

Azure A + + + + + 0 + + + + + (iXio-'M) Azure B + + + + + 0 0 + + + (iXio-'M) Azure B + + + 0 0 + + + + (SXio-*M) Azure B + + + + + 0 0 + + + + (IXIO"SM)

Key: + + + + = intensely stained; + + + = strongly stained; + + = slightly stained; + = very slightly stained; ± = stained in some, but not in all specimens; o = not stained.

TABLE 6 The effect of dyes on the birefringence of the external membrane of living A. proteus, the staining of the membrane, and the induction of pinocytosis Staining of Time Increase in external Induction of Dye Molarity birefringence membrane pinocytosis Azure A 0-005 pinkish purple Azure B OOOI pinkish Azure C at. sol. aq. o Basic fuchsin . pink ry rarely Brilliant cresyl blue purple Crystal violet. purple Dahlia . 0-0005 pinkish purple t occasionally Janus green . 0-0005 green-blue Methyl green . Methylene blue 0-0005 blue Neutral red . yellov shred Neutral violet 0-0005 red Nile blue 00005 blue Pyronine G . 0-005 orange-pink Safranin 0001 10 to 15 Thionin 0-0005 S pink t occasionally Toluidine blue 0-0005 S purple Victoria blue . 0-0005 purple-blue « occasionally

Acid fuchsin . 00005 15 to 30 Aurantia 0-005 10 tO 20 Eosin B o-ooi 15 to 30 Eosin Y o-ooi 25 Erythrosin B . 15 to 25 Fluorescein o-oooi 15 to 25 Light green . 00005 30 Methyl blue . 00005 15 to 20 Orange G 0-0005 15 to 30 Phloxine OOOI 15 Trypan blue . 0-0005 15 tO 20 Key: + + + + = striking increase in birefringence; ++ = slight increase in birefringence; + = very slight increase in birefringence; ± = possibly a very slight increase in birefringence; o = no effect; i = induces pinocytosis.