445 The vital staining of Amoeba proteus By JENNIFER M. BYRNE ...

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 

445 

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 staining 

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 organism 

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 solutions 

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 millilitres 

of each dye solution were pipetted into a solid watch glass and 30 amoebae 

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 subsequently 

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 microscope 

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)


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 unsupported 

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 negative 

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 pyronine 

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 refractive 

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 methylene 

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 specimens, 

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.


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


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 produce 

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 specimens. 

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


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 microscope 

support this view. In carefully handled, uncompressed amoebae there 

are no crystal vacuoles, but they are rapidly induced by the heat of the microscope 

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 cytoplasm. 

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


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 attachment 

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


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. 

References 

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456 

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Appendix 

TABLE I 

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

Dye 

Nile blue . 

Neutral red. 

Neutral violet 

Brilliant cresy 1 blue 

Methylene blue . 

Thionin 

Bismarck brown . 

Toluidine blue 

Azure A 

Azure B 

Azure C 

Pyronine G. 

Basic fuchsin 

Janus green 

Victoria blue 

Methyl green 

Dahlia 

Safranin 

Crystal violet 

3 X io~ G M 

+ 

± ±±±o 

o 

o 

0 

o 

o 

ot 

ot 

ot 

ol 

ol 

ol 

i X io~ 5 M 

+ t 

+ 

+-j- 

-|_ 

± 

± 0 

0 

o 

0 

ol 

ol 

ol 

Concentration 

i x io~ 4 M S x io-" M i x io~ s M 

3 X io~ 5 M 

+ 1 

+ 1 

+ 1 

+ 1 

+ 1 

+ 1 

_|_ 

+ 1 

+ t 

± o 

o 

ot 

-)- 

+ 1 

+ o 

o 

ol 

+ 0 

o 

+ 1 

ol 

o 

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.


TABLE 2 

The staining of cytoplasmic inclusions of A. proteus by basic dye solutions 

at 3 X io~ 6 M 

Dye 

Nile blue 

Neutral red . 


Brilliant cresyl blue 


Thionin 


Contents 

+ + + 

± ± 

Food vacuoles 

Fluid 

+ + + 

+ + 

± 

Spherical 

refractive 

bodies 

+ + + 

0 

0 

0 

0 

0 

0 

Crystal 

vacuoles 

0 0 0 0 + + + 

Small 

gramdes 

TABLE 3 


at 1 X 10- 5 M 

Dye 

Nile blue . 

Neutral red . 




Thionin 



Azure A 

Contents 

+ + + + 

+ + + + 

+ + + + 

+ + + 

+ + + 

4-4- 

4-4- 

± 

Food vacuoles 

Fluid 

+ + + 

4-4-4. 

4-4-4- 

4-4- 

4-4- 

4- 

4- 

+ 

± 

Spherical 

bodies 

4.4.4. 

4-4.4- 

4-4-4- 

0 

0 

0 

0 

0 

0 

Crystal 

vacuoles 

4.4. 

4-4- 

4- 

4- 

± 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

Small 

granules 

+ + + + 

4-4-4.4- 

4-4-4-4- 

0 

+ + + + 

0 

0 

0 

0 

TABLE 4 


at 3 X 10- 5 M 

Dye 

Nile blue . 

Neutral red . 




Thionin 



Azure A 

Azure B 

Food vacuoles 

Contents Fluid 

Spherical 

refractive 

bodies 

Crystal 

vacuoles 

Stnall 

gramdes 

Key: + + + + = intensely stained; + + + = strongly stained; + + = slightly stained; 

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


TABLE 5 


at 1X 10- 4 M, 5 X 10- 4 M, and 1 X 10- 3 M 

Food vacuoles 

Spherical 

Small 

Dye 

Contents Fluid bodies vacuoles granules 

Bismarck brown + + + + + 

0 

0 ± 

Azure A 

(iXio-'M) 

Azure B 

(iXio-'M) 

Azure B 

(SXio-*M) 

Azure B 

(IXIO" S M) 

+ + + 

+ + + 

+ + + 

+ + + 

+ + 

+ + 

+ + 

0 

0 

0 

0 

+ 

0 

0 

0 

+ + + + 

+ + + 

+ + + + 

+ + + + 

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 

Dye Molarity 

Azure A 

Azure B 

Azure C 

Basic fuchsin . 


Crystal violet. 

Dahlia . 

Janus green . 

Methyl green . 

Methylene blue 

Neutral red . 


Nile blue 

Pyronine G . 

Safranin 

Thionin 


Victoria blue . 

Acid fuchsin . 

Aurantia 

Eosin B 

Eosin Y 

Erythrosin B . 

Fluorescein 

Light green . 

Methyl blue . 

Orange G 

Phloxine 

Trypan blue . 

0-005 

OOOI 

at. sol. aq. 

0-0005 

0-0005 

0-0005 

0-0005 

00005 

0-005 

0001 

0-0005 

0-0005 

0-0005 

00005 

0-005 

o-ooi 

o-ooi 

o-oooi 

00005 

00005 

0-0005 

OOOI 

0-0005 

Time Increase in 

birefringence 

10 to 15 

S 

S 

15 to 30 

10 tO 20 

15 to 30 

25 

15 to 25 

15 to 25 

30 

15 to 20 

15 to 30 

15 

15 tO 20 

Staining of 

external 

membrane 

pinkish purple 

pinkish o 

pink 

purple 

purple 

pinkish purple 

green-blue 

blue 

yellov shred 

red 

blue 

orange-pink 

pink 

purple 

purple-blue 

Induction of 

pinocytosis 

ry rarely 

t occasionally 

t occasionally 

« occasionally 

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.

445 The vital staining of Amoeba proteus By JENNIFER M. BYRNE ...

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