47
The uptake of dyes by extracted phospholipids and
cerebrosides
By JENNIFER M. BYRNE
(From the Cytological Laboratory, Department of Zoology, University
Museum, Oxford)
Summary
It was found that basic dyes were without exception taken up by lecithin, cephalin, and
sphingomyelin, while the typical acid dyes were not taken up by any of the phospholipids. However, the weakly acidic dyes aurantia, eosin Y, eosin SS, and erythrosin
were taken up by all phospholipids, although to a smaller extent than the basic dyes.
Basic dyes were taken up by cerebrosides, but to a smaller extent than by the phospholipids. The basic metachromatic dyes gave a strongly metachromatic reaction with
cerebrosides. None of the acid dyes tried was taken up by cerebroside.
The direct observation that basic dyes were taken up less strongly by cerebrosides
than by phospholipids was confirmed by spectrophotometric readings.
Basic dyes were found to be insoluble in olive oil, with the exception of Bismarck
brown, neutral red, acridine orange, Nile blue, and chrysoidine. The acid dyes tried
were without exception insoluble in olive oil.
Oleic acid took up all basic dyes and some acid dyes.
It is suggested that a chemical reaction is at least in part responsible for the uptake of
basic dyes by conjugated lipids.
Introduction
IN the process of vital dyeing, basic dyes tend to colour certain pre-existing
cell inclusions—often phospholipid globules or inclusions with a phospholipid
sheath (Shafiq and Casselman, 1954; Casselman and Baker, 1955; Chou, 1957;
Malhotra, 1961). Hence it is of interest to investigate the reactions of dyes,
both acid and basic, vital and non-vital, with isolated, purified phospholipids
and cerebrosides, with the intention of applying the information obtained to
the interpretation of the mechanism of uptake of dyes by the living cell.
Some investigations have been made on the uptake of dyes by lecithin by
Overton (1900) and Seki (1933, 1956). Overton also investigated solutions
and a suspension of 'cerebrin' (crude cerebroside), but a systematic investigation of the action of dyes on phospholipids and cerebrosides does not seem to
have been carried out.
Some phospholipids, notably lecithin and cephalin, produce myelin figures
on contact with water (Virchow, 1854; Browaeys and Dervichian, 1946;
Dervichian, 1946; Stoeckenius, 1959). A preliminary investigation showed
that certain dyes were taken up from their aqueous solutions by the myelin
figures, and this seemed to afford a convenient method for the microscopical
investigation of the action of dyes on these phospholipids. Dervichian and
Magnant (1946) found that the myelin figures of lecithin could be coloured by
certain dyes, but the dyes used were not specified.
[Quarterly Journal of Microscopical Science, Vol. 103, part 1, pp. 47-56, March 1962.]
48
Byrne—The uptake of dyes by extracted
Materials and experimental methods
The phospholipids and cerebrosides were extracted from sheep's brains by
Weill's method (1930), with an additional extraction of lecithin from egg-yolk
by the application of Weill's method to eggs. Sphingomyelin was also extracted by the technique cited by Levene (1914), and cerebrosides by that of
Bodansky and Fay (1938). The crude cerebroside resulting from these extractions was further purified by treatment with ether in a Soxhlet for 24 h,
followed by two recrystallizations from alcohol/chloroform (Rosenheim, 1913)
until it was negative to the acid haematein test (Baker, 1946, 1947) and so
presumably contained little or no phospholipid impurity.
Both acid and basic, vital and non-vital dyes were used. Vital dyes are
commonly used at a concentration of about 0-02% w/v. With neutral red or
methylene blue, this concentration corresponds to a molarity of approximately
0-0007 M, and so all dye solutions were made up at this molarity. In making
up basic fuchsin equal amounts of rosanilin and para-rosanilin were assumed
to be present, and with safranin, equal amounts of di- and trimethylphenosafranin. The molecular weight of pyrrol blue is unknown and hence it was
used at an arbitrary concentration of 0-02%. Spirit-soluble eosin (ethyl eosin)
and alkali blue were used as saturated aqueous solutions, although these
correspond to a molarity less than the standard 0-0007 M. Azocarmine tends
to form a colloidal solution at low temperatures. It was therefore warmed to
50° C and used soon after it had cooled to room temperature.
A smear of lecithin or cephalin was made in the bottom of a cavity slide,
and 5 drops of the 0-0007 M dye solution added. Myelin figures were produced
quite normally in the dye solutions at this concentration. The effect of the dye
was observed microscopically. When the dye is taken up, the myelin figures
appear clearly coloured against a colourless or nearly colourless background.
The uptake of dyes was checked by removing the coverslip and immersing the
slide in distilled water. The dyes that had seemed not to have been taken up
were in most cases washed off, but in a few instances the smear retained a
slight tinge on washing. Re-examination of such a smear showed that the
colour was not in the myelin figures but trapped in the interstices between
them.
Sphingomyelin does not form myelin figures. However, it is possible to see
microscopically whether or not a dye is taken up by observation of the smear
itself. Again, 5 drops of dye solution were added to a smear made in the bottom
of a cavity slide, and if the dye were taken up the smear appeared coloured on
a colourless or nearly colourless background. Smears of sphingomyelin were
estimated to be about 25 ju thick as a general rule (maximum 40 /x).
The cerebroside that had been purified until negative to the acid haematein
test was a hard, white powder. Slides were made by mixing a few particles
of cerebroside with a little ether in a cavity slide and evaporating off the
ether. The powder remained attached to the slide. The dye was then added.
Absorption or non-absorption of the dye was easily observed.
phospholipids and cerebrosides
49
Spectrophotometric readings were taken to obtain relative figures for the
degree of uptake of dye by phospholipids and cerebrosides. The instrument
used was a Unicam S.P. 350 D.G. spectrophotometer. o-i M solutions of
lecithin, cephalin, sphingomyelin, and cerebroside in xylene were used. Five
millilitres of each were shaken with excess dye solution (1 o ml of o-1 % aqueous
basic fuchsin) and left to stand for 48 h at room temperature. Some of the
dye partitioned out and coloured the lipid solutions. The absorption maximum
of basic fuchsin does not shift in these different solutions but remains at a
wavelength of 550 m/x. All readings were taken at this wavelength. Dilution
curves of the different solutions were then plotted.
In the experiments to test the solubility of dyes in lipids, 2 ml of lipid
(olive oil, glyceryl trioleate, or oleic acid) and 2 ml of dye solution were shaken
together and left to stand for 24 h at room temperature. The lipid and aqueous
layers separated and the dye partitioned itself between them according to its
solubility in each phase.
Results
The action of dyes on phospholipids and cerebrosides
Basic dyes are without exception taken up by lecithin, cephalin, and sphingomyelin (see table 1 in the Appendix). Most of the dyes are taken up so strongly
that the dye solution appears exhausted, but azure B and particularly
pyronine G are not taken up so strongly. The myelin figures of lecithin and
cephalin continue to be produced during the course of the experiment, and
all these figures are evenly coloured by most dyes. But some dyes, particularly
those that are taken up by phospholipids so rapidly as apparently to exhaust
the dye solution within about 5 min, colour smears of lecithin and cephalin
only in localized areas. Only a few myelin figures are visible at these points,
although a search of the smear always reveals some coloured myelin figures.
If one places a smear of cephalin under the microscope and irrigates it with
dye solution, some myelin figures appear almost immediately. Dye accumulates in these and intensifies, until about 5 min after the formation of one of
these figures the colour is extremely dark and the figure collapses, giving a
patch of colour on the smear. If one adds more dye solution the whole smear
can be coloured. Hence, the patchy appearance given by some dyes would seem
to be caused by a very rapid uptake of the dye by the first-formed myelin
figures, which lowers the concentration of the dye below a certain minimum,
so that later-developed figures are unstained.
The basic metachromatic dyes, azures A and B, brilliant cresyl blue, and
toluidine blue give an interesting reaction. At the very edges of the smear,
particularly at the ends, these dyes tend to give a purplish colour. These
areas are always extremely thin, and tend to be the ones which stain first. No
myelin figures are visible at these points. This apparent metachromasy may be
due to a concentration effect (Bergeron and Singer, 1958). The main part of
the smear and the myelin figures colour blue with all these dyes.
In contrast to the basic dyes, the typical acid dyes are not taken up at all by
50
Byrne—The uptake of dyes by extracted
any of the phospholipids (see table 2). However, aurantia, eosin Y, eosin SS,
and erythrosin are taken up to some extent by all the phospholipids, although
to a smaller extent than are most of the basic dyes.
It was very difficult to determine absorption or non-absorption of certain
acid dyes (alizarin red S, haematein, pyrrol blue, and picric acid) at the standard concentrations used, and so these dyes were tried in stronger solution
(o*i %). At this concentration alizarin red S seems to be taken up very slightly
by lecithin, and picric acid by lecithin and cephalin if left for a long period
(2 h or more).
Basic dyes in general are taken up by cerebrosides (see table 1) but to a
smaller extent than by any of the phospholipids. There does not appear ever
to be a complete exhaustion of the solution. Azure B, pyronine G, and
Victoria blue are taken up extremely slightly.
The basic metachromatic dyes, azures A and B, brilliant cresyl blue, toluidine blue, and thionin, give a strongly metachromatic reaction with cerebrosides. This is presumably a true metachromasy (Baker, 1958, p. 245).
None of the acid dyes tried was taken up by cerebroside (see table 2).
Spectrophotometric observations
Spectrophotometric readings were taken to obtain relative figures for the
uptake of basic dyes by phospholipids and cerebrosides. Dilution curves of
the dyed phospholipid and cerebroside solutions are shown in fig. 1. This
shows that the phospholipids fall together as a group with regard to the
amount of dye taken up by equimolecular solutions, but that within the group,
cephalin takes up most dye and sphingomyelin least. This difference does not,
however, seem significant. The cerebrosides take up much less dye. Sphingomyelin takes up X3-65, lecithin X4/0, and cephalin X4/65 as much dye as
cerebroside.
This confirms the microscopical observation that cerebrosides take up basic
dyes less strongly than do the phospholipids.
Tests of solubility
The uptake of dyes by the phospholipids and cerebrosides might be due
either to a chemical combination—a true dyeing reaction—or to a greater
solubility of the dye in question in lipid than in the water of the aqueous dye
solution—a partition coefficient effect.
The solubility of a dye in lipid can be investigated by studying the partition
of the dye in question between its aqueous dye solution and a lipid liquid at
room temperatures. Both Overton (1900) and Seki (1933,1956) investigated the
solubility of various dyes in olive oil. It was decided to repeat this experiment.
The results with basic dyes are shown in table 1. The majority of basic dyes
were found to be insoluble in olive oil, but Bismarck brown and neutral red
(compare Seki, 1933), acridine orange, and chrysoidine seemed slightly soluble.
While most of the dye remained in the aqueous layer, the olive oil was tinged
yellow in each case. Olive oil colours red with Nile blue.
phospholipids and cerebrosides
51
The acid dyes were without exception insoluble in olive oil (see table 2).
After 48 h the lipid layer was not even tinged.
The experiment was repeated with supposedly pure glyceryl trioleate
(triolein) and with oleic acid. The results can be seen in tables 1 and 2. All
the basic dyes were taken up to some extent by triolein. Most of the acid dyes
dM
dM/2
dM/3
dM/4
dM/5
dM/6
dM/7
dM/8
dM/9
FIG. 1. Spectrophotometric readings showing the uptake of basic fuchsin by
phospholipids and cerebroside. dM = o-i M.
remained completely in the aqueous layer, but aurantia, eosin Y, eosin SS,
erythrosin, picric acid, and possibly methyl orange tinged the lipid, although
in each case most of the dye remained in the aqueous layer.
Basic dyes were again without exception taken up by oleic acid, as one would
expect (Seki, 1933, 1956). Of the acid dyes, aurantia, eosin Y, eosin SS, erythrosin, and possibly picric acid were taken up by oleic acid.
Discussion
The only phospholipid on which any work seems to have been carried out
previously is lecithin. The results of my experiments with this substance
52
Byrne—The uptake of dyes by extracted
agree in general with those of earlier workers. Both Overton (1900) and Seki
(1933) investigated the action of dyes on suspensions of lecithin and solutions
of lecithin in organic solvents, and found' that basic dyes were taken up
strongly, while acid dyes were (with a few exceptions) taken up scarcely or not
at all. Seki (1956) later used a mixture of lecithin and decalin, and found that
basic dyes were taken up by this mixture, while they were not taken up by
decalin alone. Some acid dyes were taken up more strongly by this mixture
than by decalin alone, including spirit-soluble eosin (compare with table 2).
My results agree exactly with those of Dervichian and Magnant (1946).
These workers found that the myelin figures of lecithin took up basic dyes,
while acid dyes were without effect. They obtained a similar result with
coacervates of lecithin and a weak gel. Unfortunately they did not specify
what dyes they used.
The results of the action of dyes on cephalin and sphingomyelin fit into the
same general picture.
My results with cerebroside do not agree with those of Overton (1900) on
solutions and suspensions of 'cerebrin'. It is possible that Overton's 'cerebrin'
was cerebroside heavily contaminated with phospholipid. This would account
for his finding 'cerebrin' in all ways comparable with lecithin.
The results of the investigation of the solubility of dyes in olive oil agree in
general with those of Overton (1900) and Seki(i933, 1956), although Victoria
blue does not seem to be soluble in olive oil (Seki, 1956).
Overton (1900) obtained the same results with triolein as with olive oil. My
results are in such strong conflict with his that contamination of my specimen
of triolein with fatty-acid homologues was suspected. The partition effect
between dye solutions and oleic acid seems to confirm this. All basic dyes are
taken up by both triolein and oleic acid, and it is perhaps significant that the
basic dyes can be arranged in much the same order with regard to speed and
degree of uptake in both series of experiments. Also, the same acid dyes are
taken up by both triolein and oleic acid. This similarity between oleic acid
and triolein strongly suggests that it was the presence of free oleic acid or
other fatty-acids in the triolein sample that caused the apparent solubility of
dyes in it. The differential staining of olive oil and triolein with Nile blue
confirms this (Cain, 1947).
From this it would seem that the results obtained with olive oil represent
the true solubilities of the various dyes in neutral lipid.
Overton believed the uptake of dyes by lecithin to be due to their greater
solubility in this substance than in the water of the aqueous dye solutions.
But my own results, and also those of Overton himself and of Seki, seem to
indicate that something other than solubility must be acting in the uptake of
dyes by conjugated lipids. No chemical reaction is probable between a neutral
triglyceride and a dye; but it can be seen from the results of the uptake of
dyes by oleic acid and by triolein contaminated with fatty-acid, that when
suitable reacting sites are present in the lipid, basic dyes are taken up. It
may then be suggested that a genuine chemical reaction is at least in part
phospholipids and cerebrosides
53
responsible for this uptake, and for the uptake of dyes by conjugated lipids,
which also have available reacting sites.
The phosphoric acid radicle of the phospholipids would seem to provide a
site for the reaction of basic dyes. The cerebrosides do not have this phosphoric acid group, and this may account for the difference in degree of uptake
of basic dyes by the phospholipids and cerebrosides, as revealed microscopically (see table 1), and spectrophotometrically (see fig. 1). The slight
uptake of basic dyes observed with cerebrosides may be due to reaction at
other (less strong) available sites, which are common to both cerebrosides and
phospholipids. Alternatively, a reaction between basic dyes and the carbohydrate component of cerebrosides might occur. Crystal violet and gentian
violet, for instance, are used by botanists to dye starch grains (Nemec, 1906).
But what is the mechanism of uptake of those acid dyes that colour phospholipids ? Picric acid is taken up only by lecithin and cephalin. This uptake is
extremely slight, and is evident only if a long period is allowed. Picric acid
does not colour any part of the smear that is not incorporated in a myelin
figure. Hence, considering the small size of the molecule, it seems possible
that the picrate ions merely pass into the interior of the myelin figures with the
water that contributes to their formation, without combining with the lipid
in any way. The possibility that this explanation might apply to all the dyes
that are taken up by lecithin and cephalin is ruled out by the fact that the
other dyes that are taken up stain the whole smear, including the parts that
have not produced myelin figures. Also, as mentioned before, myelin figures
that have been intensely stained with certain basic dyes tend to collapse to
form an amorphous stained mass. This suggests a combination of some sort between the dye and the lipid of the myelin figures, and not merely the presence
of the dye in the water inside the figures. In support of this, Dervichian
and Magnant (1946) have suggested that basic dyes render lecithin somewhat
insoluble. Soya lecithin, for instance, which disperses completely in water,
produces coloured myelin figures in dilute basic dye solutions.
Aurantia, eosin Y, eosin SS, and erythrosin colour all phospholipids,
including sphingomyelin, which does not produce myelin figures. These dyes
cannot then be taken up in the same manner as is picric acid. A reaction
between the dye ions and the amino-groups of the phospholipids might conceivably take place, but if this is so, it is difficult to see why the cerebrosides
are not also coloured. Experiments will be carried out to test the ability of
these acid dyes to colour cytoplasmic inclusions in living cells.
I am most grateful to Dr. J. R. Baker, F.R.S., for the help and valuable
advice given during the course of this work, and to Professor Sir A. C. Hardy,
F.R.S., for accommodating me in his Department. The work was carried out
during the tenure of a Medical Research Council Scholarship.
References
BAKER, J. R., 1946. Quart. J. micr. Sci., 87, 441.
1947. Ibid., 88, 463.
54
Byrne—The uptake of dyes by extracted
BAKER, J. R., 1958. Principles of biological microtechnique. London (Methuen).
BERGERON, J. A., and SINGER, M., 1958. J. biophys. biochem. Cytol., 4, 443.
BODANSKY, M., and FAY, M., 1938. Laboratory manual of physiological chemistry. New York
(Wiley).
BROWAEYS, J., and DERVICHIAN, D., 1946. C.R. Soc. Biol. Paris, 140, 136.
CAIN, A. J., 1947. Quart. J. micr. Sci., 88, 383.
CASSELMAN, W. G. B., and BAKER, J. R., 1955. Quart. J. micr. Sci., 96, 49.
CHOU, J. T. Y., 1957. Ibid., 98, 59.
DERVICHIAN, D., 1946. Trans. Farad. Soc, 42B, 180.
and MAGNANT, C , 1946. C.R. Soc. Biol. Paris, 140, 95.
LEVENE, P. A., 1914. J. biol. Chem., 18, 453.
MALHOTRA, S. K., 1961. Quart. J. micr. Sci., 102, 83.
NEMEC, B., 1906. Ber. dtsch. bot. Ges., 24, 528.
OVERTON, E., 1900. Jahrb. wiss. Bot., 34, 669.
ROSENHEIM, O., 1913. Biochem, J., 7, 604.
SEKI, M., 1933. Z. Zellforsch., 19, 289.
1956. Arch. Hist. Jap., 10, 601.
SHAFIQ, S. A., and CASSELMAN, W. G. B., 1954. Quart. J. micr. Sci., 95, 315.
STOECKENIUS, W., 1959. J. biophys. biochem. Cytol., 5, 491.
VIRCHOW, R., 1854. Virchows Arch., 6, 562.
WEILL, A., 1930. Arch. ges. Physiol., 223, 351.
Lecithin
Cephalin
Sphingomyelin
+ + reddish
+ + purple
+ + purple
+ + purple
+ purple
Cerebroside
o
o
o
+ yellowish
+ red
O
O
+
o
o
o
o
o
+ yellowish
O
o
O
O
Olive oil
Impure
triolein
Oleic acid
KEY. + + + + = dye taken up very strongly; solution exhausted; + + + = dye taken up strongly but solution still tinged;
+ + = dye taken up slightly; + =dye taken up extremely slightly; O = dye not taken up.
acridine orange .
azure A
azure B
basic fuchsin
Bismarck brown
brilliant cresyl blue
chrysoidine
crystal violet
dahlia
Janus green B .
methyl green
methylene blue .
neutral red
Nile blue .
pyronine G
safranin
thionin
toluidine blue .
Victoria blue
Dye
Phospholipids
The uptake of basic dyes by lipids
TABLE I
Appendix
f
acid fuchsin
alizarin red S .
.. ,, „ o - i %
alkali blue
aurantia
.
.
azocarmine
chlorazol black E
cresol red
eosin Y .
.
eosin SS
.
.
erythrosin B
.
haematein
o-i%
light green
methyl blue
methyl orange .
orange G
.
phenol red
picric acid
.,
.. ° - i %
pyrrol blue
„
.. 0 1 %
trypan blue
xylidine red
Dye
•
0
0
0
0
0
0
0
+
0
0
0
0
O
O
O
O
O
O
O
+
O
0
0
O
++
—
++
+++
+++
+++
+++
_
O
O
O
0
O
O
O
0
0
0
0
0
0
0
0
++
++
—
O
+
+O+
++
+
O
O
O
O
0
0
—
+ O+ +
+
0
O
O
—
Cephalin
Sphingomyelin
O
O
O
—
O
O
O
0
O
O
O
—
O
O
O
O
0
0
0
0
0
0
0
0
Cerebroside
O
O
O
O
O
O
O
O
O
O
O
—
O
O
O
O
O
O
O
O
O
O
O
O
Olive oil
O
O
O
O
+
+
O
O
+?
O
O
+
+
+
—
O
O
O
+
O
0
O
O
O
Impure
triolein
0
0
0
+
0
+
0
0
0
0
0
0
+
+++
+++
_
0
0
0
+++
O
O
0
0
Okie acid
= dye taken up very strongly; solution exhausted; + + + = dye taken up strongly but solution still tinged;
dye taken up slightly; + = dye taken up extremely slightly; O — dye not taken up; — — no observation.
.
.
.
.
.
.
.
.
.
O
O
Lecithin
Phospholipids
The uptake of acid dyes by lipids
TABLE 2
f
t
.to