area chosen is -J- mm. square, this will give the number which
were contained in | mm. cube of the mixture,, and multiplying
this by the number of times the blood was diluted, the result
will be the number of corpuscles in - mm. cube of blood.
Schaefer thinks that it is more convenient to have the quad-
ratic markings upon the micrometer glass of the eye-piece than
upon the slide, which is a practical point. The quadratic
markings are shown in Fig. 22. To measure any square, it is
only necessary to take the stage micrometer, ruled in milli-
metres and decimals, and adjusting the draw tube, make the
side of one square correspond exactly to an interval of | mm.
on the stage micrometer. It will then be convenient to mark
the tube at this point, and then, in all subsequent work, if the
tube be kept at this line and a slide is used of the thickness of
the micrometer and the same lens and eye -piece, the side of a
square will always be | mm. This method is the one in general
use.
Another less frequently employed is that of Malassez, which
is also described by Schaefer as follows : A little of the mixture
of blood and sulphate of soda is transferred to a very fine flat-
50
MANUAL OF HISTOLOGY.
tened capillary tube, the capacity of a given length of which has
been ascertained previously and marked on the slide to which
the tube is fixed. Thus, in his capillary tube a length of 400 mi-
cromillimetres represents the T ^V.^ P art f a cubic millimetre
of the mixture. The counting is performed with the aid of a
squared ocular micrometer, the microscope tube having been
previously so adjusted by the aid of a stage micrometer that
the side of the square shall have the value of one of the lengths
(400 n l for example) marked on the slide. The result of the
FIG. 21. Hayem and Nachet's apparatus for blood-counting.
counting gives the number of corpuscles in a known quantity
(TBT.-S c.mm.) of the mixture, and the number in a whole cubic
millimetre can therefore be readily determined.
Dr. Keyes uses a modification of the method of Hayem and
Nachet, making a dilution of 1 to 250, in order to render the
counting more easy. In Fig. 21 the pipette, A, is filled up to
the mark, 5 D ; it is then emptied into the glass vessel, F. The
pulp of the finger of the patient whose blood is to be tested
should be pierced with a triangular needle (glover's). Quick
A micromillimetre (O =
THE BLOOD.
51
but firm pressure down the finger will at once force out a drop
from the punctured spot. The blood must be drawn imme-
diately into the capillary pipette lest it coagulate. When the
pipette is full to the mark 2, its point should be rapidly wiped
clean of any blood adhering to the outside, and the contents at
once blown into the artificial serum in the cup, F. A little
suction back and forth clears the tube of any blood-corpuscles
which may have adhered to the glass within. Both tubes
should be carefully washed before being put away.
The mixture is now to be thoroughly agitated with the glass
rod, and before it has time to settle, a drop is placed in the
middle of the cell on the slide, D, care being taken that the
drop is not large enough to touch any part of the circumference
of the cell. The covering glass, E, should at once be placed
upon the cell. Should the drop be too large, so that when the
thin cover is adjusted it spreads out too much, the glass should
be cleansed and the attempt made anew. Finally, a small drop
of water or saliva is applied to the edge of the covering glass,
under which it circulates around the top of the cell, serving to
hold the cover in place and pre-
vent evaporation. The slide is
then put in position and when
the corpuscles have all settled
to the bottom of the fluid, the
counting should begin. The
following detailed plan is then
given by Dr. Keyes :
"It is better to count each
of the sixteen squares and write
down its number separately,
so that in counting the square
beneath it, should there be any
doubt about counting a given
corpuscle lying upon the line, a glance at the number recorded
for the square above may remove all doubt. Many corpuscles
will be found lying upon the outside lines bounding the large
square. I have adopted the rule of rejecting all those lying
upon the upper and right-hand outside lines (of the large
square) and counting all those lying on the lower and left-
hand outside lines.
After having thus obtained the number of red corpuscles
FIG. 22. Blood corpuscles as seen with the
squared ocular micrometer. (Keyes.)
02 MANUAL OF HISTOLOGY.
situated within the large square, it becomes easy, by a simple
equation, to find the number in a cubic millimetre. A single
count, however, exposes to sources of error, and in order to
approach more nearly to exactness, I have uniformly counted
the number contained in the large square in five different por-
tions of the field (sometimes ten), and have taken a mean of the
whole number of counts as the standard.
The computation is as follows : The glass cell on the slide
is % mm. deep. The eye-piece micrometer marks off - mm.
square, therefore the count of red corpuscles (or white, as the
case may be) must indicate the number contained (in the dilu-
tion used) in -J- mm. cube. But J mm. cube is -^ of a c.mrn.,
therefore the number counted must be multiplied by 125 ; and
the blood was diluted by adding 250 parts of fluid to 1 of blood
(2 c.mm. to 500 c.mrn.), therefore the product above obtained
must be again multiplied by 251 to get the number of corpus-
cles in a c.mm. of pure blood. Instead of multiplying twice, a
single multiplication by the product of 125 x 251, 31,375, will
give the same result."
This method should, theoretically, be absolutely accurate, but there are vari-
ous errors which will unavoidably creep in. First of all, the tubes should be
verified as to accuracy. This has been done for me at the Winchester Observa-
tory, of Yale College, by Leonard Waldo, Esq., the astronomer in charge. My
larger glass tube is slightly different in shape from the one here represented, and
is marked so that the line at ^ indicates a capacity of 500 cubic millimetres
(0.5005 grammes of distilled water at 26.4 C.). The cubical contents of the
reservoir from the point to the line i- = 0.2425 + 0.008 = 2505 grammes =
250 c.mm., approximately. Accordingly, the marks and indicate i and -J-
a cubic centimetre, within a limit of error so small as to be practically insensi-
ble. The smaller glass tube, which is capillary, is marked 2, 2, 4, and 5.
The level 5 indicates a capacity of 5 c.mm. The capacity between the pointed
extremity and 2 is 2 c.mm., less Vfn c.mm. ; the space between 2 and 2| con-
tains .55 c.mm. ; the space between 2 and 4 contains 1.45 c.mm. ; the space
between 4 and 5 contains exactly 1 c.mm. (Waldo). The determination of these
capacities was made by using distilled water, and comparing the weight, when
filled to the various levels, with the same tube after careful drying.
These estimates are given to show one of the errors which may be met
with, and that an instrument, before using, should be verified by some one who
has special means for determining capacities of this kind. My eye-piece mi-
crometer was made for me by Rogers, of Cambridge, and the entire field was
subdivided into squares, so that every portion of it may be counted without
moving the slide. My method has been practically the same as that of Dr.
K3jes, except that I prefer diluting with one thousand parts of the diluent,
THE H^EMOCHROMOMETER. 53
and use iodized serum in place of urine. The ordinary per cent, solution
of common salt in water will also answer sufficiently well.
Kecent investigations, such as those conducted by Drs. Cut-
ter and Bradford, of Boston, have established that there is
great variation in the number of globules of an individual, de-
pending on various causes, such as the locality from which the
blood is drawn, the loss of fluids, as by diarrhoea, sweating,
increased urinary secretion, etc., and even the period of the
day, week, or year. These general conclusions have also been
sustained by Hayem, of Paris, in researches which are still
being prosecuted.
When one further considers that we have no definite stand-
ard of comparison ; that the instrument is apt to be imperfect ;
that there is a liability of errors to the amount of 10 per cent.;
that skill and practice are required in manipulation, it is by no
means difficult to see that the haematometer is not calculated
at present to introduce much scientific precision into medicine,
unless the most extraordinary precautions are taken in every
case, and these all duly noted.
Blood crystals. The pigment of the blood occurs usually
in an amorphous form, and is called haematine. The brownish
red needles found in extravasated blood are known as haema-
toidine.
Haemoglobin also occurs in most mammalian blood, and is
deposited under the form of rhombic plates. It is estimated
that about 125 grammes are present in the blood of a healthy
adult.
THE HJEMOCHROMOMETER.
According to Mantegazza and others, richness in haemoglo-
bin indicates a corresponding richness in red corpuscles, and
any special depth of color in the blood may be regarded as im-
plying a certain given number of red corpuscles to the cubic
millimetre. While this ratio appears to hold true in health, it
fails in disease. Thus, a condition which we recognize as anae-
mia may be almost wholly due to a loss of haemoglobin in the
corpuscle, or an actual loss of red corpuscles, together with a
diminished amount of haemoglobin in those that remain. In
the cachexia of cancer the number of the corpuscles may be
sustained, but their haemoglobin diminished. In diabetes mel-
54: MANUAL OF HISTOLOGY.
litus, on the other hand, there may be an excess of red cor-
puscles, while there is a diminution of their haemoglobin. In
anaemia, from hemorrhage, there is an actual loss both of cor-
puscles and of haemoglobin in those that remain.
To facilitate the estimation of haemoglobin, an instrument
has been devised by Malassez and Verick (Paris), called the
TicemocTiroinometer, 1 which is easily manipulated, and bids fail-
to establish some facts of practical utility (see Archives de
Phys., 1877, p. 1).
It consists of a hand-screen, to which a movable prismatic
trough, containing a colored fluid, is attached, and a modified
Potain pipette. By means of this apparatus the richness of
the blood in haemoglobin, and the maximum quantity of oxy-
gen which it can absorb, may be determined. To use the ap-
paratus the pipette is first filled up to a certain point with the
blood to be examined, and then diluted with 100 parts of water.
The reservoir of the pipette is then filled with the diluted
blood. The screen has two holes; behind one of these the
prismatic trough is made to slide up and down, the color of the
fluid contained in it of course varying in intensity, according
to the extent of the upward and downward motion. Behind
the other opening the reservoir of the pipette is secured by
means of a little elastic ring. The screen is now held against
the light (preferably white light ; sunlight is to be especially
avoided), and the trough moved until the color of the blood
mixture is matched by its own color. Then the figure on the
scale attached to one side of the trough is read off, arid this
indicates, by reference to the table annexed to the apparatus,
the points to be determined. If the blood to be examined be
deeply colored, the aqueous blood-mixture is made in the pro-
portion of to 100 ; if it be but slightly colored, in the propor-
tion of 2 to 100.
BIBLIOGRAPHY.
WELCKER. Pragervierteljahreschr. XLIV., p. 60. 1854. Zeitschr. f. rat. Med.
3, XX., p. 280.
SCIIULTZE, MAX. Archiv f. mikrosk. Anat. I., p. 35. 1865.
KOI.LETT. Strieker's Manual of Histology. New York, 1872.
WOODWARD. Ara. Jour, of the Med. Sci., Jan., 1875. N. Y. Med. Rec., Jan. 31, 1880.
1 To be obtained of J. F. Reynders & Co., New York city.
BIBLIOGKAPHY. 55
KELSCH. Arch, de Phys. Vol. II. 1875.
KEYES. Am. Jour, of the Med. Sci., Jan., 1876.
HEITZMANN. New York Med. Jour.. April, 1877.
MANTEGAZZA. Berl. Klin. Woch., April 1, 1878.
RANVIER. Traite technique d'histologie. Paris, 1877 et seq.
HAYEM. Archives de Phys. 2 Ser., T. VI., p. 201 et seq. 1879.
BIZZOZERO, G., and SALVIOLI, G. Centralb. f. d. Med. Wiss. 16, p. 273. 1879.
POUCHET. Gaz. Med. de Paris. 14, 16. 1879.
CUTLER, E. G., and BRADFORD, E. H. Journal of Phys. Vol. I. 18781879.
BOETTCHER. Archiv f. raikrosk. Anat. Bd. XIV. p. 73. 1877.
KL'EIN and E. NOBLE SMITH. Atlas of Histology. 1879.
ELSBERG. Annals of the N. Y. Academy of Sciences. Vol. I. , Nos. 9 and 10. 1879.
(A very extensive bibliography.)
SATTERTHWAITE, T. E. Arch, of Comp. Med. N. Y. II. 1880.
BAXTER and WILLCOCK. Lancet, March 6, 13, 20, 1880.
CHAPTER IV.
EPITHELIUM.
THE skin, mucous surfaces of the body and various pas-
sages in connection with them, are evenly coated with bodies
of peculiar shape, which are united together to form a cover-
ing of one or more layers.
In some places, as upon the external portions of the epider-
mis, the corpuscles are more or less flattened. Elsewhere, as in
the ducts of secreting glands and in the trachea and fallopian
tubes, they are cylindrical, and the free extremities are often
surmounted by cilia fine, hair-like processes, which have a
vibratile movement that propels solid matters, such as sputa
and ova, in some special direction. In other parts, again, as in
the collecting tubes of the kidney, near the apices of the pyra-
mids, a cuboidal variety is found. Intermediate or transitional
forms are also frequently met with in all parts of the body.
A characteristic of epithelium which is especially note-
worthy is that the same species is not found uniformly in the
same position. Sometimes this mutation of type is governed
by the physical laws that regulate the growth and development
of the subject, or it may be a consequence of disease. An ex-
ample of the former peculiarity is to be noted in the larynx,
where the ciliated corpuscles of infancy part with their cilia
from advancing age, or indeed may become flattened.
As an example of pathological change it is not uncommon
to find villosities covered with the most beautifully marked
cylindrical epithelium, springing from the ordinary mucous
membrane, just where the superficial corpuscles happen to be
somewhat flattened in their normal state.
The use to which the part is put has also an important influ-
ence in governing the shape and other attributes of the corpus-
cles. Where they are exposed to the drying action of the air,
to harsh usage, and continued friction, as upon the hands and
EPITHELIUM. 57
feet, they become flattened, dry, and horny ; in the interior of
the body, on the other hand, where such conditions do not
exist, they are succulent and pliable.
Ordinary flattened or squamous epithelium. This is best
obtained by scraping the back of one's tongue with a blunt
instrument. The scrapings should then be mounted in equal
parts of the common salt solution (J per cent.) and glycerine.
The epithelial bodies may in this way be readily studied. They
are separate or grouped together in collections of two or more.
In diameter they vary between ^TT an & TTT incn - Tne sur ~
faces are all bevelled, and at the same time are uneven or
ridged ; consequently they overlap one another to a certain
degree, and the inequalities of one corpuscle fit into those of
another. The most superficial epithelium is the thinnest, and,
conversely, the deepest is apt to be the most nearly spheroidal.
Intermixed in the mucus will be seen the so-called mucous
or salivary corpuscles. They are not very numerous, but are de-
tected by the "molecular" or Brownian movement of their in-
terior. In size they closely resemble the white corpuscles of the
blood, but, as a rule, exhibit no amoaboid motion ; the white glob-
ules, on the other hand, rarely have any Brownian movement.
The surfaces of the epithelia are often so covered with bac-
teria that they are only recognized with some difficulty. These
little bodies are wonderfully uniform in size, and are disposed
in the most regular manner. Looking straight down upon
them they appear to be minute spheres with a diameter aver-
aging between ^QT and ^TUTTF incn - Closer inspection and
examination of the corpuscles at their free edges shows that
the bacteria are in reality rod-shaped, and that they adhere to
the corpuscles by their extremities, standing in such cases
vertical to the surface. A high power, such as the immersion
T V, develops this point quite clearly.
Incidentally the mucin of the mucus may be seen to advan-
tage in the scrapings of the mouth or tongue. To a drop or two
add another drop of commercial alcohol and a drop of the or-
dinary hsematoxylin solution. The alcohol will coagulate the
mucin, which then takes the form of filaments and branching
networks ; the logwood will make them distinctly visible.
Epithelium from the skin may be studied in one of two
methods. Take a fresh specimen from the palmar surface of
the hand or plantar of the foot, freeze it in a section cutter,
58 MANUAL OF HISTOLOGY.
take off a thin slice with a knife, immerse for a few seconds in
a dilute solution of acetic acid (J per cent.), and then mount in
glycerine and water ; or a similar portion of the skin may be
steeped in a weak, sherry-colored, watery solution of the bichro-
mate of potassium (gr. ij. iij. f. j.) for several days and then
hardened in alcohol, first of 80 per cent., then of 90 per cent.,
finally of 95 per cent, strength ; this latter process taking several
days, and ending when the specimen is thoroughly hard. Sec-
tions may then be made in the usual way. By the use of acetic
acid the nuclei will readily be seen in the lower strata of the epi-
dermis, while the outermost layers have none, or, at least, none
that can be demonstrated by the usual histological methods.
Three different strata can now be recognized : 1, the stra-
tum corneum, or corneous layer, in which the corpuscles are
flattened, and appear to have no nuclei ; 2, the rete mucosum,
or malpighian layer, immediately underlying the former, and
composed of cuboidal elements, armed with spines or prickles,
as they are often called ; lastly, 3, there is the pigmented layer,
which overlies the papillae. The bodies of the latter corpuscles
are infiltrated with particles of melanine, which is the cause of
the dark color in the skin of the negro and swarthy races.
Maceration of the epidermis in liquor potassse is an excellent
method for exhibiting the individual elements ; after a few min-
utes they will swell up and detach themselves from one another.
It was thought, until quite recently, that these prickle cells interdigitate
with one another, but Ranvier has claimed that they are continuous with those
of adjacent corpuscles (see chapter on the Skin). This point is difficult to set-
tle, as it requires a special method and lenses of high power. Ranvier injected
a one-fourth per cent, solution of osmic acid into the lower layers of the epider-
mis, using a hypodermic syringe, and driving the fluid right and left.
There is a form of flattened and pigmented epithelium that
may be seen by examining the external surface of the choroid,
the ciliary processes, and the posterior surface of the iris. In
the choroid these bodies look like a mosaic of polyhedral cells.
Such specimens may be permanently preserved by simply dry-
ing them, and then mounting in dammar or Canada balsam.
Ciliated epithelium. The movement of living cilia is readily
seen. All that is necessary is to take the common frog (Rana
temporaria), draw out his tongue, and then observing the teat-
like projections at the posterior part, snip one off.
NC
MEDICAL SCHOUL LiumA
EPITHELIUM. 59
This little piece is then to be mounted in a one-fourth per
cent, salt solution, or serum, and examined. Along the free
edge of the mucous membrane the cilia will be seen engaged in
active vibratile motion. The appearance presented by a broad
expanse of moving cilia has been aptly described as resembling
a field of grain which is being swept by the wind, though the
motion is often much more rapid than this comparison would
imply. It will be seen that various substances, such as blood
globules, are propelled in a definite direction. When the frog's
mouth is open, all solid particles that are lodged upon the
mucous membrane are carried quietly but inevitably toward
the gullet, and down toward the stomach. The power of the
ciliary movement may be estimated, in a measure, by placing
some light but adhering body upon the anterior portion of the
roof of the mouth, and then inverting the animal. The sub-
stance immediately begins to ascend against gravity, and soon
is wedged in the gullet. The same force, though acting in an
opposite direction, expels mucus, pus, and indeed all solid
matters, from the cavities of the human lungs ; it also propels
the ova through the Fallopian tubes into the uterus. In ex-
cessive catarrh from mucous membranes the epithelial bodies
may themselves be expelled, so that they are not infrequently
found with their cilia attached, as in the nasal discharges. After
death cilia are hard to recognize ; they contract down to little
knobs on the surface of the cells, and can only be demonstrated
when the eye looks directly down upon them. Osmic acid is
useful to preserve them in their natural condition. Take a fresh
specimen and immerse it for twenty-four hours in a one-fourth
percent, osmic acid solution, and for another twenty -four hours
in dilute alcohol ; then tease and mount in glycerine and water.
It will be observed that each cilium is a slim, straight rod, which
is apparently structureless ; they rest upon a band, which, with
a high power, may be seen to have vertical striations.
Effect of reagents. By making use of the moist chamber
(Fig. 19, p. 42), and placing a drop of chloroform in the cor-
ner of the cell, it will be seen that the action of the cilia rap-
idly stops, while, if the chloroform be removed, it will again
resume its activity.
If carbonic acid gas is admitted, the action of the cilia will
at first be accelerated, but subsequently retarded, and eventu-
ally stopped (Kuehne).
60 MANUAL OF HISTOLOGY.
After shutting off the carbonic acid gas and admitting oxy-
gen, the action will again commence. When the ordinary
motion has ceased, the gradual application of heat will cause
it to return ; but if the temperature be raised continuously, a
point will soon be reached where the excessive heat will cause
the motion again to stop.
Columnar or cylindrical epithelium. This is the epithe-
lium^ar excellence of the digestive tract, clothing the mucous
membrane from the cardiac orifice of the stomach to the anus.
It is also found at the orifices of the ducts of the large excretory
glands, such as the liver and pancreas, in the milk-passages of
the nipple, and in some parts of the generative system. These
cells are tall and narrow, standing vertical to the surface of the
mucous membrane. Sometimes they are broadest at their free
extremity, at other times about the middle, so that when viewed
from above they appear to be separated from one another. The
nuclei are rounded, and are either placed about the middle of
the cell or near the attached border. They admit of consider-
able variation, however, as to size and shape, some of those in
immediate contact being broad at one extremity, and some
broad at the others ; the free edge also may be uneven.
Scrape the surface of a frog's tongue or a rabbit's intestine
after washing ; the cells will be seen to advantage. Place
some of the scrapings in a drop of glycerine and water to
which another drop of dilute acetic acid (J per cent.) has been
added, and mount. In this way the nuclei will be brought
clearly into view. The cells closely resemble in their shape
the columnar variety, except that they have no cilia. Among
them will almost always be found chalice or goblet cells. They
lie among the columnar corpuscles, and are usually shorter,
but broader, expanding in the centre, and terminating at their
attached extremities in a single or double process. The sur-
face is cupped. They contain one or more nuclei ; whether
they are a distinctive cell or not is as yet uncertain. Some
suppose them to be the ordinary columnar cell undergoing
mucoid degeneration ; others that they are not epithelial at