salts of the dye bases, for instance, rosanilin acetate) form
combinations with acid dyes (_i.e._ salts of the dye acids, for
instance, ammonium picrate) which are to be regarded as neutral dyes,
such as rosanilin picrate. Their employment offers considerable
difficulties as they are very imperfectly soluble in water. A practical
application of them was first possible after Ehrlich had ascertained
that certain series of the neutral dyes are easily soluble in excess of
the acid dye, and so the preparation of solutions of the required
strength, readily kept, was made possible. Among the basic dyes which
are suitable for this purpose are those particularly which contain the
ammonium group, especially methyl green, methylene blue, amethyst
violet[5] (tetraethylsafraninchloride), and to a certain extent pyronin
and rhodamin also. In contradistinction to these, the members of the
triphenylmethan series, such as fuchsin, methyl violet, bismarck brown,
phosphin, indazine, are in general less suited for the purpose, with the
exception of methyl green already mentioned. The acid dyes specially
suited for the production of soluble neutral stains are the easily
soluble salts of the polysulpho-acids. The salts of the carbonyl acids
and other acid phenol dyes are but little suitable: and least of all,
the nitro dyes. Specially to be mentioned among the acid dye series are
those which can be used for the preparation of the neutral mixtures:
orange g., acid fuchsin, narcein (an easy soluble yellow dye, the sodium
salt of sulphanilic acid--hydrazo-[beta]-naphtholsulphonic acid).
If a solution of methyl green be allowed to fall drop by drop into a
solution of an acid dye, for instance orange g., a coarse precipitate
first results, which dissolves completely on the further addition of the
orange. No more orange should be added than is necessary for complete
solution. This is the type of a simple neutral staining fluid.
Chemically the above-mentioned example may be thus explained; in this
mixture all three basic groups of the methyl green are united with the
acid dye, so that we have produced a triacid compound of methyl green.
Simple neutral mixtures, which have one constituent in common, may be
combined together straight away. This is very important for triple
staining, which can only be attained by mixing together two simple
neutral mixtures, each consisting of two components. A chemical
decomposition need not be feared. We thus get mixtures containing three
and more colours. Theoretically there are two possibilities for such
combinations:
1. Staining mixtures of 1 acid and 2 basic dyes,
_e.g._ orange--amethyst--methyl green;
narcein--pyronin--methyl green;
narcein--pyronin--methylene blue.
2. Staining mixtures of 2 acids and 1 base, in particular the mixture to
be described later in detail of
orange g.--acid fuchsin--methyl green.
Further narcein--acid fuchsin--methyl green,
and the corresponding combinations with methylene blue, and amethyst
violet may be mentioned.
The importance of these neutral staining solutions lies in the fact that
they pick out definite substances, which would not be demonstrated by
the individual components, and which we therefore call =neutrophil=.
Elements which have an affinity for basic dyes, such as nuclear
substances, stain in these neutral mixtures purely in the colour of the
basic dye; acidophil elements in that of one of the two acid dyes;
whilst those portions of tissue which from their constitution have an
equal affinity for acid and basic dyes, attract the neutral compound, as
such, and therefore stain in the mixed colour.
* * * * *
The eosine-methylene blue mixtures are exceptional in so far, that it is
possible with them, for a short time at least, to preserve active
solutions, in which with an excess of basic methylene blue, enough eosin
is dissolved for both to come into play. A drawback however of such
mixtures is, that in them precipitates are very easily produced, which
render the preparation quite useless. This danger is particularly great
in freshly prepared solutions. In solutions, such as Chenzinsky's, which
can be kept active for a longer time, it is less. Hence fresh solutions
stain far more intensely and more variously than older ones, and are
therefore used in special cases (see page 46). If the stain is
successful the appearances are very instructive. Nuclei are blue,
haemoglobin red, neutrophil granulation violet, acidophil pure red, mast
cell granulation deep blue, forming one of the most beautiful
microscopic pictures.
For practical purposes, besides the iodine and iodine-eosine solution
described below (see page 46) the following are especially used:
1. =Haematoxylin solution with eosin or orange g.=
Eosin (cryst.) 0.5
Haematoxylin 2.0
Alcohol abs.
Aqu. dest.
Glycerine _aa_ 100.0
Glacial acetic acid 10.0
Alum in excess
The fluid must stand for some weeks. The preparations, fixed in absolute
alcohol, or by short heating, stain in from half-an-hour to two hours.
The haemoglobin and eosinophil granules are red, the nuclei stain in the
colour of haematoxylin. The solution must be very carefully washed off.
2. In the practical application of the triacid fluid, particular care
must be taken, as M. Heidenhain first shewed, that the dyes are
=chemically pure=[6]. Formerly granules, apparently basophil, were
frequently observed in the white blood corpuscles, particularly in the
region of the nucleus. They were not recognised, even by practised
observers (_e.g._ Neusser) as artificial, but were regarded as
preformed, and were described as perinuclear forms. Since the employment
of pure dyes these appearances, whose meaning for a long time puzzled
us, are but seldom seen.
Saturated watery solutions of the three dyes are first prepared, and
cleared by standing for some considerable time. The following mixture is
now made:
13-14 c.c. Orange-g. solution
6-7 c.c. Acid fuchsin solution
15 c.c. Aqu. dest.
15 c.c. Alcohol
12.5 c.c. Methyl green
10 c.c. Alcohol
10 c.c. Glycerine
These fluids are measured in the above-mentioned order, with the same
measuring glass; and from the addition of methyl green onwards the fluid
is thoroughly shaken. The solution can be used at once, and keeps
indefinitely. The staining of the blood specimen in triacid requires
only a little fixation, cp. page 35. The stain is completed in five
minutes at most.
The nuclei are greenish, the red blood corpuscles orange, the acidophil
granulation copper red, the neutrophil violet. The mast cells stand out
by "negative staining" as peculiar bright, almost white cells, with
nuclei of a pale green colour.
The triacid stain is very convenient. It is much to be recommended for
good general preparations; =it is indispensable in all cases where the
study of the neutrophil granulations is concerned=.
3. =Basic double staining.= Saturated, watery methyl-green solution is
mixed with alcoholic fuchsin.
The stain, which only requires a small fixation, is completed in a few
minutes, and colours the nuclei green, the red blood corpuscles red, the
protoplasm of the leucocytes fuchsin colour. It is therefore specially
suited for demonstration preparations of lymphatic leukaemia.
4. Eosin-methylene blue mixtures, for example Chenzinsky's fluid:
Concentrated watery methylene blue solution 40 c.c.
1/2% eosin solution in 70% alcohol 20 c.c.
Aqua dest. 40 c.c.
This fluid is fairly stable, but must always be filtered before use. It
only requires a fixation of the specimen for five minutes in absolute
alcohol. The staining takes 6-24 hours (in air-tight watch-glasses) at
blood temperature. The nuclei and the mast cell granulations stain deep
blue, malaria plasmodia light sky blue, red corpuscles and eosinophil
granules a fine red.
This solution is particularly suited for the study of the nuclei, the
baso and eosinophil granulations, and it is used by preference for
anaemic blood, and also for lymphatic leukaemia.
5. 10 c.c. of a 1 per cent. watery eosin solution, with 8 c.c. methylal,
and 10 c.c. of a saturated watery solution of methylene blue are mixed,
and used at once, see page 41. Time of staining 1, at most 2 minutes.
The staining is characteristic only in preparations very carefully fixed
by heat. The mast cell granulations are stained pure blue, the
eosinophil red, the neutrophil in mixed colour.
6. Jenner's stain consists of a solution in methyl alcohol of the
precipitate formed by adding eosine to methylene blue.
Grubler's water soluble eosine, yellow 1.25% } a.a. watery
" medicinal, methylene blue 1% } solutions.
Precipitate allowed to stand 24 hours, and then dried at 55 deg.. It is then
made up to 1/2% in methyl alcohol (Merck). The stain may be obtained
from R. Kanthack, 18, Berners Street, London, ready for use. It is
exceedingly sensitive to acids and alkalis. Fixation is effected by
heat. Time of staining 1-4 minutes.
Before we pass to the histology of the blood, two important methods may
be described, for which the dried blood preparation is employed
directly, without previous fixation: 1. the recognition of glycogen in
the blood; 2. the microscopic test of the distribution of the alkali of
the blood.
1. _Recognition of glycogen in blood._
This may be effected in two ways. The original procedure consisted in
putting the preparation into a drop of thick cleared iodine-indiarubber
solution under the microscope, as had been already recommended by
Ehrlich for the recognition of glycogen.
The following method is still better. The preparation is placed in a
closed vessel containing iodine crystals. Within a few minutes it takes
on a dark brown colour, and is then mounted in a saturated laevulose
solution, whose index of refraction is very high. To preserve these
specimens they must be surrounded with some kind of cover-glass cement.
By the use of better methods the red blood corpuscles which have taken
on the iodine stain stand out, without having undergone any
morphological change. The white blood corpuscles are only slightly
stained. All parts containing glycogen on the contrary, whether the
glycogen be in the white blood corpuscles, or extracellular, are
characterised by a beautiful mahogany brown colour. The second
modification of this method is specially to be recommended on account of
the strong clearing action of the laevulose syrup. In using the
iodine-indiarubber solution a small quantity of glycogen in the cells
may escape observation owing to the opaqueness of the indiarubber, and
occasionally too by the separate staining of the same. The second more
delicate method is for this reason recommended, in the investigation of
cases of diabetes and other diseases[7].
2. The microscopic test of the distribution of alkali in the blood.
These methods rest on a procedure of Mylius for the estimation of the
amount of alkali in glass. Iodine-eosine is a red compound easily
soluble in water, which is not soluble in ether, chloroform, or toluol.
But the free coloured acid, which is precipitated by acidifying
solutions of the salt, is very sparingly soluble in water. It is, on the
contrary, very easily soluble in organic solvents, so that by shaking,
it completely passes over into an etherial solution, which becomes
yellow. If this solution be allowed to fall on glass, on which deposits
of alkali have been formed by decomposition, they stand out in a fine
red colour as the result of the production of the deeply coloured salt.
In its application to the blood, of course the vessels used for staining
as well as the cover-glasses must be cleaned from all adhering traces of
alkali by means of acids. The dry specimen is thrown directly after its
preparation into a glass vessel containing a chloroform or
chloroform-toluol solution of free iodine-eosine. In a short time it
becomes dark red. It is then quickly transferred to another vessel
containing pure chloroform, which is once more changed, and the
preparation still wet from the chloroform is then mounted in canada
balsam. In such preparations the morphological elements have preserved
their shape completely. The plasma shews a distinct red colour, whilst
the red corpuscles have taken up no colour. The protoplasm of the white
corpuscles is red, the nuclei appear as spaces, because unstained
(=negative nuclear staining=). The disintegrated corpuscles and the fibrin
which is produced, shew an intense red stain. These stains are
peculiarly instructive, and shew many details which are not visible in
other methods. The study of these preparations is really of the highest
value, since they allow the products of manipulation of the dry
preparation and every error of production to stand out in the most
reliable manner, and so render possible a kind of automatic control. The
scientific value of this method lies in the fact that it throws light on
the distribution of the alkali in the individual elements of the blood.
It appears that free alkali reacting on iodine-eosine is not present in
the nuclei; these must therefore have a neutral or an acid reaction. On
the contrary the protoplasm of the leucocytes is always alkaline, and
the largest amount of alkali is held by the protoplasm of the
lymphocytes. We call particular attention, in this connection, to the
strong alkalinity of the blood platelets.
FOOTNOTES:
[4] Kloenne and Mueller, Berlin, supply these after Ehrlich's directions.
[5] Baden Anilin and Soda manufactory, Kalle and Co.
[6] At M. Heidenhain's instigation, the Anilin-dye Company of Berlin
have prepared the three dyes in the crystalline form.
[7] It may also be used for the recognition of glycogen in secretions.
For instance, gonorrhoeal pus always shews a considerable glycogen
reaction of the pus cells. It is found, moreover, in cells which
originate from tumours, whether these be present in exudations, or
obtained by scraping.
B. NORMAL AND PATHOLOGICAL HISTOLOGY OF THE BLOOD.
In satisfactorily prepared dry specimens =the red blood corpuscles= keep
their natural size and shape, and their biconcavity is plainly seen.
They present a distinct round homogeneous form, of about 7.5 mu in
diameter. They are most intensely coloured in a broad peripheral layer,
and most faintly in the centre corresponding to their depression. With
all stains mentioned above the stroma is quite uncoloured, and the
haemoglobin exclusively attracts the stain, so that for a practised
observer the depth of stain gives a certain indication of the haemoglobin
equivalent of each cell, and a better one than the natural colour of the
haemoglobin in the fresh specimen. Corpuscles poor in haemoglobin are
easily recognised by their fainter staining, especially by the still
greater brightness of the central zone. When somewhat more marked, they
present appearances which from the isolated staining of the periphery
Litten has happily named "pessary" forms. The faint staining of a red
corpuscle cannot be explained, as E. Grawitz assumes, by a diminished
affinity of the haemoglobin for the dye. Qualitative changes of this kind
of the haemoglobin, expressing themselves in an altered relationship
towards dyes, do not occur, even in anaemic blood. If in the latter, the
blood discs stain less intensely, this is due exclusively to the smaller
amount of haemoglobin.
A diminution in the haemoglobin content can in this way be shewn in all
anaemic conditions, especially in posthaemorrhagic, secondary and
chlorotic cases. On the contrary, as Laache first observed, in the
pernicious anaemias, the haemoglobin equivalent of the individual discs is
raised.
To appreciate correctly pathological conditions, it must always be borne
in mind, that in normal blood the individual red blood corpuscles are by
no means of the same value. Step by step some of the cells are used up
and replaced by new. Every drop of blood contains, side by side, the
most various stages of life of fully formed erythrocytes. For this
reason influences which affect the blood--provided their intensity does
not exceed a certain degree--cannot equally influence all red
corpuscles. The least resistant elements, that is, the oldest, will
succumb to the effect of influences, to which other and more vigorous
cells adapt themselves.
To influences, of this moderate degree, belongs without doubt the anaemic
constitution of the blood as such, the effect of which in this direction
one can best investigate in cases of posthaemorrhagic anaemia.
In all anaemic conditions we observe characteristic changes in the blood
discs.
A. =Anaemic or polychromatophil degeneration.=
This change in the red blood corpuscles, first described by Ehrlich, to
which the second name was given later by Gabritschewski, is =only
recognisable in stained preparations=. The red blood discs, which under
normal circumstances stain in pure haemoglobin colour, now take on a
mixed colour. For instance, the red corpuscles are pure red in
preparations of normal blood, stained with haematoxylin-eosine mixture.
But in preparations of blood of a chromic anaemia stained with the same
solution, in which possibly all stages of the degeneration in question
are present, one sees red discs with a faint inclination to violet;
others which are bluish red; and at the end of the series, forms stained
a fairly intense blue, in which scarcely a trace of red can be seen, and
which by their peculiar notched periphery are evidently to be regarded
as dying elements.
Ehrlich put forward the theory, that this remarkable behaviour towards
dyes indicates a gradual death of the red blood corpuscles, that is of
the old forms, leading to a coagulation necrosis of the discoplasm. The
latter takes up, as is the case in coagulation necrosis, the proteids of
the blood, and acquires thereby the power of combining with nuclear
stains. At the same time the discoplasm loses its power of retaining the
haemoglobin, and gives it up to the blood plasma in ever increasing
quantity as the change proceeds. Hence the disc continues to lose the
capacity for the specific haemoglobin stain.
Objection has been raised to these views from many quarters, especially
from Gabritschewski, and afterwards from Askanazy, Dunin and others. The
polychromatophil discs are not, they say, dying forms, but on the
contrary represent young individuals. The circumstance, that in certain
anaemias the early stages of the nucleated red corpuscles are variously
polychromatic, was evidence for this opinion.
In view of the great theoretical importance which attaches to this
subject, the grounds for regarding this change as degenerative, may be
here shortly brought forward.
1. The appearance of the erythrocytes which shew polychromatophilia in
the highest degree. By the notching of their margins they appear to eyes
practised in the judgment of morphological conditions, in a stage almost
of dissolution, and as well-pronounced degeneration forms.
2. The fact that by animal experiment, for instance, in inanition, their
appearance in large numbers in the blood can be produced. That is,
precisely in conditions, where there can be least question of a fresh
production of red blood corpuscles.
3. The clinical experience, that in acute losses of blood in man, these
staining anomalies, can be observed in numerous cells, within so short a
time as the first 24 hours. Whilst in our observations, which are very
numerous upon this point, embracing several hundred cases, and carried
out with particular care, no nucleated red blood corpuscles in this
space of time can be found in man[8].
4. The polychromatophil degeneration can frequently be observed in
nucleated red blood corpuscles, particularly in the megaloblasts. This
fact can be so easily established that it can hardly escape even an
unpractised observer, and it was sufficiently familiar to Ehrlich, who
first directed attention to these conditions. The fact that the
normoblasts, which are typical of normal regeneration, are as a rule
free from polychromatophil degeneration, gave the key for the
interpretation of this appearance. And similarly for the nucleated red
blood corpuscles of lower animals. Askanazy asserts that the nucleated
red blood corpuscles of the bone-marrow, which he was able to
investigate in a case of empyema, shew, immediately after the resection
of the ribs, complete polychromatophilia. This perhaps depends on the
peculiarities of the case, or on the uncertainty of the staining method:
eosine-methylene blue stain, which is for this purpose very unreliable,
since slight overstaining towards blue readily occurs. (We expressly
advise the use of the triacid solution or of the haematoxylin-eosine
mixture for the study of the anaemic degenerations.)
After what has been adduced, we hold in agreement with the recent work
of Pappenheim, and Maragliano, that the appearance of polychromatophilia
is a sign of degeneration. To explain the presence of erythroblasts
which have undergone these changes we must suppose that in severe
injuries to the life of the blood these elements are not produced in the
usual fashion, but from the very beginning are morbidly altered.
Analogies from general pathology suggest themselves in sufficient
number.
B. A second change that we find in the red blood corpuscles of the
anaemias, is =poikilocytosis=.
By this name a change of the blood is denoted, where along with normal
red blood corpuscles, larger, smaller and minute red elements are found
in greater or less number. The excessively large cells are found in
pernicious anaemia, as Laache first observed, and as has since been
generally confirmed. On the contrary in all other severe or moderate
anaemic conditions, the red corpuscles shew a diminution in volume, and
in their amount of haemoglobin. This contradiction, which Laache first
mentioned, but was unable to explain, has found a satisfactory solution
in Ehrlich's researches on the nucleated precursors of the myelocytes
and normocytes (see below).
The blood picture of the anaemias is made still more complicated in that
the diminutive cells do not preserve their normal shape, but assume the
well-known irregular forms: pear-, balloon-, saucer-, canoe-shapes.
Nevertheless in good dry preparations the smallest forms usually still
shew the central depression. The so-called "microcytes" constitute an
exception to this statement. These are small round forms, to which was
allotted in the early days of the microscopic investigation of the
blood, a special significance for the severe anaemias. They are however
nothing but contraction forms of the poikilocytes, as the crenated are
of the normal corpuscles. Consequently microcytes are but seldom found
in dried specimens, whilst in wet preparations they are easily seen
after some time. It is further of importance to know, that in fresh
blood the poikilocytes exhibit certain movements, which have already
given rise to mistakes in many ways. Thus at one time the poikilocytes
were considered to be the cause of malaria. More recently the somewhat
larger sizes were regarded by Klebs, Perles as amoebae and similar
organisms. In agreement with Hayem, who from the very first described
these forms as =pseudo-parasites=, a warning must be given against
attributing a parasitic character to them.
The origin of poikilocytosis, previously the subject of much
discussion, is now generally explained in Ehrlich's way. For the mere
fact, that by careful heating, poikilocytosis can be experimentally
produced in any blood, forces one to the assumption that the
poikilocytes are products of a fragmentation of the red blood corpuscles
("schistocytes," Ehrlich). And correspondingly the smallest fragments
shew the biconcave form in the dry specimen; for they too contain the
specific protoplasm of the disc "which possesses the inherent tendency
to assume the typical biconcave form in a state of equilibrium."