FROM 
PROCEEDINGS 
OF THE 
American Society of Microscopists. 
FIFTEENTH ANNUAL MEETING, 
HELD AT 
ROCHESTER, N. Y. 
1892. AMERICAN MICROSCOPICAL SOCIETY. 
63 
MICROSCOPICAL RESEARCHES OF THE CORPUSCULAR 
ELEMENTS OF BLOOD. 
M. L- Holbrook, M. D., New York City. 
I. 
The Structure of the Red Blood Corpuscles of Man. 
Although since the time of the discovery of the blood corpuscles, 
over 200 years ago, a good many observations- have been made by 
various microscopists, certain questions still remain unsettled. One 
of these questions is, Are the red blood corpuscles only structureless 
formations, of certain chemical compounds, or are they endowed 
with properties of life? A second question is, What is the structure 
of the colorless corpuscles of the blood, and in what relation do 
they stand to the constitution ? A third question is, What is the 
source and significance of the so-called third element of the blood, 
the blood plaques, to the presence of which attention has recently 
been called? I shall in this paper consider only the red blood 
corpuscles, their structure, their varying appearance under the 
microscope, and their relationship to other protoplasmic forma- 
tions. The other questions I must postpone to some future occa- 
sion, because my studies in them have not been completed. 
It seems to me plain that the living organism, being traversed by 
a closed vascular system, cannot carry only chemical compounds 
destitute of life, as morphological elements. The liquid portion 
unquestionably is a carrier of such compounds, holding a good deal 
of albumen, fibrine, and certain salts; carrying also the nourishing 
matter destined for nutrition; also effete materials that are destined 
to be eliminated by the secretory organs. The corpuscles, on the 
contrary, serve as the carriers of a complex and highly oxidized 
material, which we call haemoglobin. Obviously this haemoglobin, 
important as it is, is destitute of life ; whereas the bearers of it, or 64 
PROCEEDINGS OF THE 
the red blood corpuscles, must be alive and subject to changes in 
form and in other properties which we find only in living matter. 
In our present views of the animal organism we consider the so- 
called protoplasm as the real seat of life. It has been shown, how- 
ever, that protoplasm is by no means structureless; that what has 
previously been termed granules of the protoplasm are really the 
points of intersection of a minute reticulum traversing it. This 
reticulum has been shown to be the contractile or living mater 
proper; whereas in the meshes of this reticulum a liquid is present 
which, though varying in consistency and chemical compositon, is 
acknowledged to hold nitrogen and to serve as the carrier of both 
nourishing and effete materials. It has been shown that the animal 
organism is a continuous mass of protoplasm, continuous through- 
out all the tissues, the consistency of which depends upon the chem- 
ical changes of the liquid, semi-solid, or solid filling material of the 
meshes ; whereas the reticulum of the living matter proper is not 
only continuous, but also identical throughout all the tissues of the 
organism, both in animals and in plants. 
The main mass of the animal body, aside from its fluid portion, 
is the so-called connective tissue, which, by a varying consistency 
of its basis substance, furnishes the organism with shape and firm- 
ness. The skeleton of the vertebrates is a variety of connective 
tissue, mainly destined to build up the frame. Dense and highly 
elastic formations of connective tissue are represented by the dif- 
ferent varieties of cartilage covering the articular ends of the bone, 
giving firmness to air passages, to the frame of the nose, the orifice 
of the ear,etc. Highly elastic representatives of fibrous connective 
tissue are the tendons, ligaments, the fascia, aponeurosis, and the 
derma of the skin. More delicate varieties of fibrous connective tissue 
accompany and hold together all the formations of higher grades 
of vitality, such as the muscle fibers and the nerves. The connective 
tissues we know to be simply supporters of the blood and lymph 
vessels. Neither muscle nor nerve fibers do as such contain blood 
or lymph vessels. It is invariably the accompanying connective 
tissue that holds such formations; yet the muscle fibers are endowed, 
so to say, with far more vitality than the connective tissue, since 
they serve for motor purposes. We know that the living or con- 
tractile matter is contained in the muscles in large quantities; that 
the constituent, primitive, sarcous elements, though considerably 
larger than the granules of ordinary protoplasm, are identical with 
the latter, and are interconnected throughout the motor apparatus AMERICAN MICROSCOPICAL SOCIETY. 
65 
by delicate threads. The central formation of the nervous system, 
the gray substance and its offshoots, the axis cylinders are again 
reticulated in structure, but far more delicate than in the muscles, 
evidently fitting them for rapid contractions of the reticulum, con- 
veying sensory and motor impulses with great rapidity through the 
organism. The epithelial tissue is likewise destitute of blood and 
lymph vessels, but owing to the presence of the horny filling mate- 
rial of the meshes endowed with a high degree of resistance, such as 
we observe in the scarf skin, the hairs, the crystalline lens. The 
epithelia performs all the secretory work of the body through the 
material carried into them by the accompanying blood vessels located 
in fibrous connective tissue. The real structure of the epithelia is 
the same as that of protoplasm in general, although the chemical 
function is specifically that of secretory and excreting effete mate- 
rial. 
In blood we have only a liquid, the so-called serum or plasma, in 
which there are present large numbers of red and smaller numbers 
of colorless corpuscles, all of which are single, independent indi- 
viduals rolling about in the serum, and, as far as the red blood cor- 
puscles are concerned, serving as the carriers of oxidizing and nour- 
ishing materials. This liquid, according to our present views, is 
certainly not entitled to the name of a tissue. In every amseba we 
occasionally observe vacuoles containing a liquid in which we see 
detached granules of living matter. The vascular system of the 
amaeba-that is, the vacuoles-is obviously homologous to the closed 
vascular system of higher animals, the only difference being that the 
vacuoles are transient, the blood vessels permanent formations. The 
granules of living matter suspended in vacuoles are probably homol- 
ogous with the blood corpuscles in the blood of higher animals. 
In order to examine fresh blood under the microscope we must 
keep it in a moist chamber to prevent the evaporation of its fluid 
parts for some length of time. To secure this we oil the edges of a 
thin covering glass with any oil, sweet-oil, cod-liver oil, castor-oil, or 
vaseline ; glycerine is unfit, since it is soluble in water. For apply- 
ing the oil use a little roll of paper in the form of a very small rod. 
The skin, best of the palm of the left thumb, is pricked with a sharp 
needle, rendered aseptic by heating it in the flame of a spirit-lamp. 
The drop of blood should be small; therefore the prick need not be 
deep. The painful sensation is reduced to a minimum by firm com- 
pression of the parts to be pricked with the finger and thumb of the 66 
PROCEEDINGS OF THE 
operator's left hand. A slight alternate pressure or squeezing and 
relaxing will in a few seconds secure a droplet of blood, the size of 
which should not exceed that of a medium-sized pin-head. The 
slide should be applied at once over the droplet. A rotatory move- 
ment of the slide serves to spread it evenly, and immediately the 
cover, which has been prepared in advance and so placed that it 
may be quickly seized, is put over it. Its oiled edges should her- 
metically seal in the blood and prevent evaporation. The bleeding 
from the prick soon ceases, and in order to avoid infection, which 
would rarely happen, we may apply to the wound a little dilute 
acetic acid or a weak solution of sublimate, or even hot water. 
Some practice is required to get just sufficient oil on the edges of 
the cover. Too little allows the penetration of air from the outside, 
thus rendering the specimen unfit for observation. Too much causes 
a concentration of the blood and an overcrowding of the corpuscles, 
which prevents their individual observation, and also mixes oil 
globules with the blood serum. If the specimen be properly pre- 
pared it remains fit for study for about fourten days. Such a pro- 
tracted study is the special feature of my procedure and the result is 
my present paper. I have examined specimens of blood from about 
thirty persons, of varying ages and constitutional characteristics, 
including both sexes. The youngest was a tuberculous child four 
years old, the oldest a woman of seventy-six. 
The drop of blood after mounting should be at once transferred 
to the stage of the microscope and studied with a good immersion 
lens (water or cedar oil) of an amplifying power of at least 1,000 or 
1,200. My studies were made in C. Heitzmanh's laboratory, with a 
^--inch immersion lens made by the late Tolles, of Boston, which, 
as shown by comparison, I have found superior to any other immer- 
sion lens, not excluding the celebrated ones by Zeiss, of Jena. 
The first thing which strikes the student is the great variety in 
the tint of the red blood corpuscles. The deepest tint I found in 
the blood of a Vassar girl, twenty-six years old, whose anthropo- 
metric measurements gave the largest respiratory capacity in a class 
of five hundred girls. Another very brilliant specimen was in a 
woman seventy years old, of remarkably strong body and vigorous 
health, and withal an abstainer from flesh food. The palest tint 
was seen in the blood of an Irish servant girl, about twenty-five 
years of age, who, although strong and healthy before coming to 
our shores five years ago, became pale and chlorotic here, as so 
many of these girls do, on account of their changed and unhygienic AMERICAN MICROSCOPICAL SOCIETY. 
67 
habits of eating, dress, and absence of outdoor active life. All 
grades between these extremes were observed in other subjects. A 
peculiar lake color was noticed in the red blood corpuscles of a 
girl, twenty years old, who had just passed an attack of parotitis 
(mumps). A pronounced dusky lake color was seen in the blood 
corpuscles of a man, thirty-five years old, whose spleen was notice- 
ably enlarged. A still more pronounced hazy lake color was seen 
in the red corpuscles of a physician, forty-two years of age, who a 
few months after the observation died with sarcoma of the lungs ; 
and still another case of a deep lake color was observed in a woman, 
over fifty years of age, whose kidneys were in a cirrhotic condition. 
A second striking feature under the microscope was the variation 
in the coin-like arrangement of the red blood corpuscles. This was 
found invariably in the blood of persons of a good constitution, less 
frequently in the blood of individuals of a midling constitution, and 
almost absent in persons of a very poor constitution, especially those 
who had tuberculosis, chlorosis, or who were broken down by acute 
or chronic ailments. 
In one instance observation extending over years showed a tem- 
porary presence and absence of these coin-like rolls. It is quite 
probable that the agglutination of the red blood corpuscles is due 
to the presence of fibrine in the serum of the blood. 
A third striking feature was the appearance of crenations along 
the borders of the corpuscles immediately after their transference to 
the microscope, which in most instances required not more than one 
minute after the prick of the skin. The crenations were most 
numerous and conspicuous on the red blood corpuscles of persons 
of an excellent constitution, with a pronounced red color. 
It has been said that these creifetions are caused by alcoholic drink, 
but in one specimen from a man with a fine constitution nearly every 
one was crenated, and I can testify to his being absolutely temperate. 
Only the smallest sized corpuscles, termed since Hagen, 1876, 
haematoblasts, never become crenated, though in one case I did 
observe very delicate thorn-like projections from their margin. This 
was in the case of a broken-down man, about sixty years old, with 
evidence of anaemia, in which the haematoblasts existed in an abnor- 
mal number. I estimated that they amounted to about one-fourth 
of the total number of corpuscles. 
The chlorotic girl before mentioned had no crenated red blood 
corpuscles upon immediate examination, but three to four hours 
later a limited number were slightly crenated. After forty-eight 68 
PROCEEDINGS OF THE 
hours there were still more. A very large number showed odd and 
unusual forms. The same girl, after a month at the sea-side, had 
far less unusual forms of corpuscles and one-third or more were 
crenated soon after being put on the slide. I have found, as a 
general rule, that the deeper the red color of the corpuscles or, in 
other words, the more highly oxidized the haemoglobin the sooner 
the crenations appear and the more numerous they are. On the 
contrary, the paler the red color of the corpuscles the later will the 
crenations take place and the scantier and more irregular they are. 
The fewest I have seen was in the red blood corpuscles of the tuber- 
culous four-year-old child quoted above. 
The crenations after several days, usually four to six days, change 
their character. Instead of showing knobbed or globular protuber- 
ances, they become more delicate and thread-like. On the sixth 
and eighth day they are mostly gone, except in these cases where 
they appear late, and their contours are, in the majority of corpus- 
cles, even and regular. The knob and thread-like prolongations 
along the periphery of the corpuscles appear in the shape of delicate 
globules of a somewhat higher refraction on the surface of the disk 
being looked at from a top view. I emphasize this feature since it 
has misled some observers. For instance, Hueter, of Germany, took 
the knobs on the surface of the disk for micro-organisms held in 
their interior. I would discard every blood corpuscle with serrated 
edges for the study of its interior structure, since this is rendered 
deceptive by the presence of knobs and folds or wrinkles which re- 
semble the reticulum and have misled some observers, as already 
stated. 
The next question arises, What causes the crenation of the red 
blood corpuscles? Elsberg was the discoverer of the reticular struc- 
ture of these formations.* 
His observations were made on blood treated with a solution of 
bichromate of potash in distilled water, diluted so as to make about 
a five per cent, solution. He saw shortly after the application of 
the preparation varying changes of the corpuscles by indentations 
and by protrusion, aside from vacuation of the flattened-out disks, 
and came to the conclusion that these changes of form were due to 
the contraction of the reticulum prevading the body of the corpus- 
cles. Such a contraction of a regularly arranged reticulum causes 
*The structure of the colored blood corpuscles. Annals of New York 
Academy of Sciences, 1879. AMERICAN MICROSCOPICAL SOCIETY. 
69 
bulging of the portion of the wall of the corpuscle between the net 
corresponding to a mesh filled with haemoglobin. Elsberg main- 
tained that this reticulum is living or contractile matter proper, the 
same as in any other protoplasmic body. The meshes, on the con- 
trary, instead of holding a nitrogenous liquid, such as in ordinary 
protoplasm, holds the chemical compound termed haemoglobin. All 
that is done by the solution of bichromate of potash is the extraction 
of the haemoglobin from the meshes of the reticulum, whereby this 
latter is rendered plainly visible. According to Elsberg, therefore, 
a red blood corpuscle is nothing but a lump of protoplasm holding 
in its meshes a liquid substance termed haemoglobin, otherwise 
endowed with all the properties of life which are generally attribu- 
table to protoplasm. 
Elsberg's great discovery has met with incredulity and unjust 
criticism. Weichselbaum, of Vienna, objects to the value of the 
discovery on the ground that the solution of bichromate of potash 
has altered and mutilated the structure of the corpuscle, though he 
ought to have known that this substance and also chromic acid in 
weak solution is admitted by all histologists to be the best preserva- 
tive of tissues-in fact, directly proven under the microscope some 
thirty years ago by Alexander Rollett. Mueller's preserving fluid 
is composed of one per cent, of bichromate of potash and two per 
cent, of sulphate of soda, and is still largely in use for the preserva- 
tion of the most delicate tissues, such as the brain, the spinal cord, 
the eye-ball, especially the retina. L. Ranvier, of Paris, who is 
considered the best French histologist, could not satisfy himself as 
to the presence of a reticulum in the interior of the red blood cor- 
puscles, but considers Elsberg's figures as foldings on the surface of 
the corpuscles. Only quite recently Dr. Alfred C. Stokes, of Tren- 
ton, New Jersey,* has, however, enthusiastically and in strong lan- 
guage confirmed Elsberg's observations point by point. I am also 
able to confirm them by a method which I hope is beyond question, 
and this method is to simply observe the same mounted droplet of 
blood up to the eighth or tenth day, prepared as previously de- 
scribed. By closely watching the red blood corpuscles we notice a 
gradual fading of these bodies, with a simultaneous gradual coloring 
of the serum in which the corpuscles are suspended. Since no re- 
agent was used in my specimens, the conclusion I draw is that the 
serum gradually dissolves the haemoglobin from the corpuscles, thus 
*Tlie Microscope, vol. 12, No. 2. 70 
AMERICAN MICROSCOPICAL SOCIETY. 
rendering its interior structure visible. Even corpuscles of a deep 
color will, a few days after mounting, become colorless or nearly so, 
and the reticulum in their interior will come plainly into view, the 
meshes being light after the extraction of the haemoglobin, and the 
reticulum gray or greenish-gray. This observation is the more re- 
liable, as, at the period of time mentioned, all crenations and folds 
on the surface disappear. Comparison of corpuscles treated accord- 
ing to the method of Elsberg with my own method compels me to 
say that the reticulum may be a trifle more conspicuous or regular 
in specimens treated with the solution of potash than in specimens 
not treated at all, which is my own method ; but there is not the 
slightest difficulty to a trained eye armed with a good immersion 
lens to see the reticulum or structure in untouched specimens, nor 
of convincing one's self of its identity with that of Elsberg. 
Beginning from the twelfth day and much earlier in blood from 
sick patients, especially with tuberculosis, the corpuscles begin to 
break asunder into granules, the reticular connections of which still 
remain visible in many cases. 
At the same time other corpuscles may become so faded that only 
faint, ghost-like shadows remain. About the fourteenth day after 
mounting the blood the specimen is, owing to these changes, ren- 
dered unfit for further observation, the serum having by this time 
assumed a reddish-yellow tint, owing to the haemoglobin being dif- 
fused through it. 
In the meantime other remarkable changes have taken place, 
which throw light upon the origin and significance of the so-called 
third element of the blood. The description of these occurrences 
I defer to some future time.