nence is usually seen (Fig. 13). The protoplasm is the sub-
stance of which the disk is made ; it has a light yellow color,
and is dull or pellucid in appearance, much like semi-solid
jelly.

Brownian and amoeboid movements. Using the same
method of preparation the white corpuscles or leucocytes are
seen to good advantage. They are much smaller than the red
disks (in the frog the reverse of human blood), and there is
wide range in size, one histologist (Klein) having described as
many as thirty varieties. In the interior, little dark spots are
sometimes seen in constant vibration. By a skilled observer they
are readily detected, even with a good \ inch glass. When
such specks are numerous the bodies are said to be granular.
In the newt's blood this phenomenon is usually best seen.
The word granule has been , applied in these cases from the
notion once prevalent that the little dots were molecules sus-
pended in a menstruum of some sort that filled the corpuscle.
This subject is now eliciting much study, but the movement,
whatever its significance may be, is called the Brownian move-
ment.

Klein, who states that the newt's leucocyte is traversed by an intracellular
network, believes that the movement just described is due to the motion of the
"disintegrated network" under the stimulus of imbibed water. Under this
explanation the oscillatory movement in the corpuscles of the human saliva
would indicate death rather than life. When fluid has been withdrawn by
evaporation the phenomenon ceases. According to other histologists this
vibratile motion is an indication of vital action.

The remarkable change in form which these corpuscles un-
dergo is a more positive indication of vital power in the leuco-
cyte. When the little body is placed under conditions which
imitate those of its natural state it commences to put forth
processes and then withdraw them, carrying on these move-
ments slowly, but with a certain degree of regularity. While
this is being accomplished the corpuscle is observed to move
about from place to place.



40



MANUAL OF HISTOLOGY.




FIG. 15. Leucocytes: a, putting out pro-
>, having withdrawn them. (Rollett.)



In Fig. 15 the leucocytes are seen. Those marked with the
letter a are engaged in amoeboid motion. The one marked b
i? in a state of contraction. This phenomenon is called amoe-
boid movement, because it resem-
bles that of the amoeba the lit-
tle microscopic organism found
in stagnant water. In order to
permit these changes to continue
for some length of time, it is well
to paint a little oil or glycerine
around the edge of the circle.
Evaporation is thus prevented.

If the warm slide be used the
changes will follow with greater
rapidity. Both Brownian and

amoeboid movements are usually confined to a limited number
of the corpuscles, and the former often to only a small portion
of the interior.

The slide * for heating consists of an ordinary glass slide
(Fig. 16) upon which is riveted a thin copper plate (b) perfor-
ated in the centre, so as to allow space for the drop of blood
which is to be examined. From the copper plate extends an
arm (c) over which is slipped a spiral copper wire (e), which is
heated by the flame of an alcohol lamp. By this means the
glass plate is kept warm and with it the drop of blood. In
order to secure a proper
amount of heat and no
more, it is customary
to put a little bit of
cocoa butter upon the
corner of the slide. The
butter melts at the tem-
perature of the body,
and after this point has
been reached the lamp
should be carried along
the wire away from the slide until the precise distance is found
at which this particular degree of heat will be maintained.
Action of a dilute salt solution. It is often difficult, and,




FIG. 16. Slide for heating : o, slide; &, copper plate ; c, arm
over which the spiral wire (d) is slipped



1 Made by T. H. McAllister, 49 Nassau Street, New York City.



THE BLOOD. 41

indeed, impossible, to obtain aqueous humor or even an animal
Huid such as has been described, and microscopists have accord-
ingly made use of a substitute that can be prepared at any time
and kept indefinitely. This is a solution of common salt in
distilled water (1 400). Add a drop of fresh frog's blood to a
drop of the salt solution, mix them well, and it will be seen
that the delicate protoplasm of the red blood-corpuscle, most
susceptible of change, is not altered in appearance, though the
body itself will change in form from the elliptical to the spher-
ical. This salt solution has been found, in practice, an excellent
substitute for blood-serum, and is very generally used in ex-
amining fresh specimens, where it is important to avoid any
material change in the corpuscle.

Action of distilled water Irrigation. The effect of water
is also noteworthy, as it is a very important consideration in
both histological and pathological work, especially the latter.
Take a drop of frog's blood, add to it an equal quantity of
distilled water and apply a cover. The nucleus or central body
will now be readily seen, surrounded by a yellow border ; the
body of the corpuscle or peripheral part will at the same time
gradually become paler and larger. Now add distilled water
slowly, drop by drop, in the following way : Take a long strip
of tissue or filter paper about half the length of the slide and in
breadth equal to one-half the diameter of the cover. Apply
the water with an ordinary minim dropper, close to the edge of
the cover, on the side opposite to the paper strip. This latter
will now take up the excess of water and cause a stream to
pass through the specimen. This process is called irrigation.
Push the paper a short distance under the edge of the cover,
and the solid particles in the fluid will be carried to the edge
of the paper, where they will remain at rest and may be ob-
served at one' s leisure.

This plan is often useful in other sorts of microscopic work, as in looking
for renal casts, urinary crystals, etc. It may save much valuable time. I first
learned it from my friend, Dr. Edward Curtis, of this city.

Continued addition of water will cause the corpuscles to
swell and after a time burst, or, at any rate, become so expand-
ed that they can scarcely be seen. When water is applied
slowly to human blood, the corpuscles soon begin to lose their



MANUAL OF HISTOLOGY.



disk-like form and assume a spheroidal, perhaps spherical con-
tour. The coloring matter then escapes, in most instances, and
they become quite transparent (see Fig. 17). Such corpuscles are
often seen in human urine where they appear as colorless rings.
In frog's or newt's blood the body of the disk first imbibes the




FIG. 17. Human red blood-globules :
a, with haemoglobin ; 6, without it. (Rol-

l3tt.)




FIG. 18. Red corpuscles of the frog
that have imbibed water. (Rollett.)



water ; later, the nucleus, which then has a sharply defined
outline. Sometimes the material of which the body is largely
composed (haemoglobin) is gathered about the nucleus, sending
off radiating prolongations to the periphery, while the imbibed
fluid is stored in the intervening spaces (see Fig. 18).

Action of carbonic acid gas. This experiment requires a
special apparatus. First of all it is essential to have a moist
chamber (Fig. 19).

Take a small, flat bit of wood about 1J inch wide, 3 inches
long, and f inch thick ; make a square opening in the centre,
sufficiently large to admit an ordinary f inch cover-glass ; this
is to be pressed to the bottom and firmly fixed, thus making a

shallow well with a glass
bottom. Into this cham-
ber are admitted, through
side holes, glass tubes (one
on each side), so that air
or gases can be carried into the chamber. When in use, the
chamber is kept moist by a drop of water, which is put in
one corner of the well, while the specimen of blood to be ex-
amined is dropped upon a large glass cover, and the latter in-
verted over the mouth of the well. In determining the effect
of carbonic acid gas upon animal life, we have merely to con-
nect the gas-chamber just described with a jar in which carbonic




FIG. 19. Moist chamber.



THE BLOOD. 43

acid gas is generated. Fig. 19 illustrates a gas or moist cham-
ber of the same general character, and devised by Dr. J. H.
Hunt, of Brooklyn. Take a large gallon flask, fill it partly
full of pulverized marble-dust, attach it by means of a rubber
tube through a perforated stopper to a Wolff's bottle, which
latter must be connected with the moist chamber. Now gener-
ate the carbonic acid gas in the flask by pouring muriatic acid
upon the marble-dust. When the gas is being evolved it will
be known by the ebullition of the water in the Wolff's bottle.
Now place the moist chamber upon the stage of the micro-
scope. Take a drop of newt's blood, dilute it with serum or an
indifferent fluid, and mount it upon a glass cover, which invert
over the well, first seeing that the edge of the cover is oiled, so
that it will remain in place. Now connect the tube of the moist
chamber with the tube of the gas-generator, and the carbonic
acid gas will enter and pass through the chamber. The rapidity
with which the current moves may be regulated by a spring
clip. As soon as the gas enters, the central body or nucleus
becomes distinctly visible, and is surrounded by a yellow halo ;
when, however, the gas is withdrawn and atmospheric air
is admitted, the nucleus and colored zone disappear. This
double experiment may be repeated a number of times. Finally
a point will be reached where all action will cease. This cen-
tral body, under such circumstances, has been called the zooid,
and the corpuscles proper the oikoid (Bruecke).

Action of acids upon the blood. Acetic acid is commonly
used in observing the changes that are produced by an acid
solution.

Take the ordinary dilute watery solution of acetic acid
(1 per cent.) so much used in laboratories, add a drop of it to
an equal amount of frog's blood. The red globules instantly
exhibit nuclei. The colorless globules also cease their motion,
if any has existed, and they become granular and shrivelled.
The term granular is used merely in a relative sense and has no
special reference to granules whether present or not, but merely
to an appearance that has already been explained.

These phenomena are more marked if the solution is con-
centrated. The red bodies, also, in such case, are apt to crack
and split up. A good way of determining the proper strength
for the ordinary acetic acid solution is to pour a little into an
ordinary watch-glass, and then add chemically pure acetic



44 MANUAL OF HISTOLOGY.

acid drop by drop until the solution is faintly acid to the
taste.

Action of alkalies upon the Hood. Take a drop of the
newt's blood and mount it in a drop of serum or of salt solu-
tion. Then, affixing a strip of bibulous paper in the way that
has been described, add drop by drop a weak solution of aqua
ammonite. A similar strip of paper, somewhat larger in size,
upon the other side, will cause a current and carry the corpus-
cles to the side of the field where the paper strip is largest, and
there the corpuscles may be observed at rest, and the altera-
tions effected by the alkali duly noted. It will be seen that
after a little time the corpuscles, both red and colorless, will
swell up and finally, after a time, provided the alkali be in
sufficient amount, disappear or become so expanded as to be
invisible. Sometimes they will burst, leaving the field evenly
stained with a homogeneous glutinous-looking substance.

Action of electricity. It seems to make little difference, so
far as the microscope is concerned, whether the continuous or
interrupted current is employed, as in either case the phe-
nomena observed are the same in quality. Take bits of tin-
foil and attach them to an ordinary glass slide, in such a
way that they are just - inch distant from one another. The
pieces of foil should be triangular in shape and have their
pointed extremities turned to one another. The specimen
should be a drop of newt's blood diluted with an equal amount
of serum, both perfectly fresh. They should be intimately
mixed with a glass rod.

Depositing a drop of this solution upon a cover-glass, it
should be inverted and placed upon the slide in such a wa}^
that it occupies an intermediate position between the bits of
tin-foil. The ordinary stage clips of the microscope are then
to be used in holding the slide firmly ih position and to press
upon the tin-foil. The only remaining task is the attaching of
conducting wires from the electrical instrument, one to each
clip. The bits of tin-foil are easily fastened to the slide ; they
have merely to be hammered out flat, when they will adhere by
simple pressure. Sometimes it may be desirable to approxi-
mate the poles. In such cases it is necessary to use two fine
bits of platinum wire. They should be flattened, and shaped
like the letter S. Rest them upon the bits of tin-foil, opposite
to one another and at the required distance apart. The cover-




THE BLOOD. 45

glass should press on them. Some little mechanical dexterity
is required to get them in position, and they are apt, after using,
to become so charged that their action upon the corpuscles
commences before they are connected with the battery. The
phenomena at the negative pole are those of an acid ; at the
positive, those of an alkali. At a distance from the line of the
current, secondary changes occur of a less regular character.

Harting has devised an apparatus which is somewhat more
elaborate, but in principle the same.

Other changes in the red corpuscles. If a drop of blood be
taken from the finger, by pricking with a needle (the triangu-
lar or glover's is the best), it will be seen
after a time that the exterior of the corpus-
cle is indented or crenated, as this change
is called. It is well shown in Fig. 20.

Examination of the circulation in the
web of a frog's foot. Take a medium-sized
frog and curarize him by injecting beneath Plo 2 o -Human
the skin, with an ordinary hypodermic syr- d t v*** crenated - < R
inge, two drops of a weak solution of curara
(12,000 in water) or a few minims of a 50 per cent, solution
of chloral hydrate (Schaefer). After a variable time the ani-
mal will be completely paralyzed, but the circulation will go
on as before.



There are many difficulties in the use of curara, depending on the variable
strength of the drug, the idiosyncrasies of the animal, and other causes that
we do not appear to understand. A solution which will produce a proper
amount of paralysis in a frog on one day will rapidly kill another frog the next
day. To ensure any reliability of action, it is well to have a specimen of
which the strength has been properly tested. Then, if time enough is at one's
disposal, a weak solution, such as the above, may be injected every hour until
the symptoms of the drug are apparent. If the subsequent recovery of the
animal is not of vital importance, the amount may be increased, for the circu-
lation will often be well shown, even if the animal does not eventually survive.

My friend, Dr. W. H. Welch, who is in charge of the Histological Labora-
tory at the Bellevue Medical College, employs a watery solution of curara.
He keeps on hand a | per cent, solution of the drug ( 1 gramme to 200 c.c. of
distilled water), and then dilutes it as occasion may warrant to i per cent., or
even -^ per cent. (1500 or 11,000). Of this diluted solution he injects four
or five drops into the dorsal lymph- sac of the frog. A still more dilute solu-
tion he is often in the habit of using, so that the frog does not come under the
influence of the drug for an hour or an hour and a half. After twenty-four to



46 MANUAL OF HISTOLOGY.

forty-eight hours the animals entirely recover, but if a stronger solution is
used, he finds the results are frequently fatal, though the animals may survive
long enough to permit a ready demonstration of the circulation, emigration of
leucocytes, etc.

Now envelop Ms body in a damp cloth and extend him upon
a cork plate about a quarter inch thick and large enough to
support the entire body. Make a small opening in the cork,
and over it place the web of the frog's foot, fastening the latter
by ordinary pins.

The circulation may in this way be studied at one' s leisure.
The red and white blood-corpuscles are seen in the arteries,
veins, and capillaries. While the red bodies pass rapidly
through the central portions of the vessels, the white creep
slowly along the walls, altering their shape as they meet with
any obstruction. Where, however, a small artery divides, it
will sometimes be seen that the corpuscles, especially the red,
are caught at the bifurcation ; parb bending to go down one
branch, and part down the other ; taking, in fact, the shape of
a saddle-bag. Such a phenomenon exhibits the elastic and
distensile properties of the corpuscle. Apply an irritant, such
as a weak solution of nitrate of silver, and after prolonged and
careful watching, the gradual exit of both white and red cor-
puscles may be seen. This procedure requires extreme pa-
tience and a co-operation of peculiarly fortunate conditions,
which are not likely to favor the beginner in microscopy.

Internal structure of the red corpuscles. As yet the inti-
mate structure of blood-corpuscles is a matter little understood,
though an abundance of theories are rife about it. Klein main-
tains that these corpuscles, in common with others in the body,
are traversed by an intracellular network. In the red cor-
puscles of the newt, especially, he says there is a network of
fibrils, with an interfibrillar hyaline ground substance, both
together forming the so-called stroma. The nucleus contains a
network of fibrils in connection with the network of the cor-
puscle proper ; the haemoglobin, a colored fluid, is contained in
the substance of the meshes of the network of the corpuscle
proper. Drs. Cutter, of Boston, and Heitzmann, of this city,
also state that there is an intracellular network. The former
regards it as due to the mycelium of a parasitic growth.

Dr. Elsberg, of this city, also states that he finds a reticu-
lar appearance after using a solution of the bichromate of



THE BLOOD. 47

potash (30 per cent, to 50 per cent, of a saturated solution in
water).

Real granules are often present in the corpuscles, as may be
proved by adding water in large quantity. They will then
become greatly distended, and bursting, the granules will be
scattered throughout the field.

If finely ground vermilion is sprinkled in the liquid, some
of the white corpuscles will take up the granules, perhaps with-
out losing their amoeboid character ; finally, they may eject
them after a longer or shorter sojourn.

According to Boettcher, the human red blood-corpuscle has a nucleus. He
exhibits it in the following way : Taking a saturated solution of corrosive subli-
mate in alcohol (96), he diffuses about fifty volumes with one of blood. The
corpuscles are deprived of their hsematin, but at the same time are preserved.
The mixture is frequently agitated, but in about twenty-four hours it is allowed
to subside, when the superincumbent fluid is poured off and alcohol added.
By further agitation for another twenty-four hours the corpuscles are thoroughly
washed, and then settle at the bottom of the vessel. Prof. Boettcher claims
in this way to have found three classes of red globules. The first are homo-
geneous and shiny throughout ; the second are clear externally, but granular
within ; the third variety exhibit a nucleus and nucleolus.

Development of ike Hood-corpuscles. In early foetal life
all the corpuscles are colorless (Klein). According to Balfour
and Foster, both colored and colorless corpuscles, at least in
the chick, are developed from solid sprouts of protoplasm, de-
rived from the middle germinal layer. There seems good rea-
son, however, to believe that the leucocytes are formed in part,
at least, from the lymphatic glands, and Klein thinks that
they are thrown off from the "germinating buds" of serous
membranes. Later, the red ones make their appearance, and
for a time are nucleated. The investigations of Neumann and
Bizzozero, showing that the red corpuscles in the medulla of
bones are also nucleated, favors the theory that bone-marrow is
one of the theatres for such corpuscular metamorphosis.

According to Hayem the production of red corpuscles in the
blood is accomplished through the agency of hcematoblasts,
i.e., minute red corpuscles. In convalescence from acute fe-
vers, or after a considerable loss of blood, these smaller bodies
may be observed in the blood for a variable time, even some
weeks.



48 MANUAL OF HISTOLOGY.

According to Recklinghausen, the colorless corpuscles may be generated
from the red corpuscles, but it is probable that they may be formed in the tis-
sues at many points, and the connective substances through their intimate asso-
ciation with the lymphatics are capable of manufacturing them in almost any
quantity. Neither of the two varieties of corpuscles, the red or the white, have
a cell-wall or outer investing membrane that can be demonstrated, though it is
not unlikely that the outer layer of protoplasm has greater density than the
more internal portions.

Wliite or colorless blood- corpuscles. The white blood-cor-
puscle is much larger, on an average, in the human species,
than the red. It is rounded in form, and is estimated as varying
between .0077 and .0120 mm. The average is .0091 mm. (Frey).
In contour they are apt to be more or less rough, and exhibit
processes. In some of these corpuscles the nucleus is distinct,
though when quite fresh a nucleus is rarely seen. If the eye
of the observer can watch the corpuscle when it is upon a
heated stage and under suitable conditions, its division may
be seen. The number contained in the system is variable, as
we shall see, depending upon a great number of conditions.

The personal observations of the author do not incline him
to regard the network which has attracted so much attention
of late years as satisfactorily shown to exist in living corpus-
cles, although there is no question but that it has been seen in
corpuscles after exposure to chemical reagents.

According to Dr. Richard Norris, there is, in mammals, a third corpuscular
element which is usually invisible and of the same size as the red ones. Some
doubt is thrown upon his alleged discovery, by the fact that the method he
employs is likely to produce artificial appearances, and therefore leads to the
supposition that the alleged bodies were merely red corpuscles decolorized.

Mode of counting the blood-corpuscles. Thanks to the
instruments of Malassez, Hayem and Nachet, and Gowers, we
are in a position to count the red blood-corpuscles with a fair
degree of accuracy.

The methods are somewhat different, but are not difficult to
understand.

Schaefer describes his plans as follows : In order to separate
the corpuscles and prevent coagulation, the blood used is first
diluted to a definite extent say a hundred times with a 10
per cent, solution of sulphate of soda. The mixing can be per-
formed in a measuring-glass if the blood is in sufficient quan-



THE BLOOD. 49

tity, but if only a small drop is obtainable, such, for example,
as is got by pricking the linger, a mixer is better. This con-
sists of a capillary tube terminating in a bulb, the capacity of
the bulb between the marks 1 and 101 being exactly 100 times
that of the tube from its point to the mark 1. A small glass
ball is inclosed in the bulb, and serves, by its movements, to
facilitate the mixing. The capillary tube is allowed to fill with
blood as far as the mark 1 ; sulphate of soda solution is then
sucked up as far as the mark 101. As it passes in, it of course
pushes the blood before it into the bulb, and the two are there
thoroughly mixed by gentle agitation.

The next thing is to count the corpuscles in a known quan-
tity of the mixture. The most convenient plan is that of
Hayem and Nachet. A slide is used, having a glass ring -J- mm.
in depth, cemented on to its upper surface. A drop of the
mixture, but not enough to fill the cell so formed, is placed in
the middle of the ring, and a perfectly flat cover-glass is so laid
on that the drop touches and adheres to it without reaching
the sides of the cell. The slide is placed on the microscope,
and as soon as the corpuscles have settled down to the bottom
of the drop, the number in a definite area is counted. If the