445
T h e 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 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
M O S T 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, p p . 445-58, 1963.]
2421.4
Gg
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
food vacuole
spherical
refractive
mitochondrion
•acuole
small
granules
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)
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 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.
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 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
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 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
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 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
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.
References
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Appendix
TABLE I
The action of basic dye solutions at various molarities on A. proteus
Concentration
Dye
Nile blue .
Neutral red.
Neutral violet
Brilliant cresy1 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 i X io~ 5 M 3 X io~ 5 M i x io~4M
+
±
±
±
±
o
+t
+
+
+
-j-|_
o
o
±
±0
0
0
o
o
ot
ot
ot
ol
ol
ol
o
0
+1
+1
+1
+1
+1
+1
__|
+1
+t
-)-
±
o
o
ot
+1
+
o
o
ol
S x io-" M i x io~ s M
+
+1
0
ol
o
o
ol
ol
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
Nile blue
Neutral red .
Neutral violet
Brilliant cresyl blue
Methylene blue .
Thionin
Bismarck brown .
Contents
Spherical
refractive
bodies
+++
+++
+++
±
±
±
±
0
++
±
±
±
±
0
0
0
0
0
Crystal
vacuoles
0 0 0 0 ++ +
Fluid
Food vacuoles
Dye
Small
gramdes
0
0
0
0
0
0
TABLE 3
The staining of cytoplasmic inclusions of A. proteus by basic dye solutions
at 1 X 10- 5 M
Food vacuoles
Spherical
Crystal
vacuoles
Small
granules
Dye
Contents
Fluid
bodies
Nile blue .
Neutral red .
Neutral violet
Brilliant cresyl blue
Methylene blue .
Thionin
Bismarck brown .
Toluidine blue
Azure A
++++
++++
++++
+++
+++
+++
4.4.4.
4-4.44-4-4-
4-44-4-
44-
0
0
0
0
±
±
+
0
0
0
±
0
0
0
4-4-4.
4-4-44-44-4-
0
0
4.4.
4-444-
±0
++++
4-4-4.44-4-4-40
+ +0 + +
TABLE 4
The staining of cytoplasmic inclusions of A. proteus by basic dye solutions
at 3 X 10- 5 M
Food vacuoles
Dye
Contents
Fluid
Spherical
refractive
bodies
Crystal
vacuoles
Stnall
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
vacuoles
Small
granules
0
0
±
++
0
+
++
0
0
+++
0
0
++++
0
0
++++
Contents
Fluid
bodies
Bismarck brown
+++
++
Azure A
+++
Dye
++++
(iXio-'M)
Azure B
(iXio-'M)
Azure B
(SXio-*M)
Azure B
(IXIO" S M)
+++
+++
+++
++
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
Time
Dye
Azure A
Azure B
Azure C
Basic fuchsin .
Molarity
Increase in
birefringence
Staining of
external
membrane
Induction of
pinocytosis
pinkish purple
pinkish
o
pink
0-005
OOOI
at. sol. aq.
ry rarely
Brilliant cresyl blue
Crystal violet.
Dahlia .
Janus green .
Methyl green .
Methylene blue
Neutral red .
Neutral violet
Nile blue
Pyronine G .
Safranin
Thionin
Toluidine blue
Victoria blue .
Acid fuchsin .
Aurantia
Eosin B
Eosin Y
Erythrosin B .
Fluorescein
Light green .
Methyl blue .
Orange G
Phloxine
Trypan blue .
purple
purple
pinkish purple
green-blue
0-0005
0-0005
blue
yellov shred
red
blue
orange-pink
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
t occasionally
10 to 15
S
S
pink
purple
purple-blue
t occasionally
« occasionally
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
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.