COLUMBIA LIBRARIES OFFSITE

Hh Al IM Sr.lf NCf S SIANDAMO

HX64098656
QP44 .B892 University college c

S I

n\A

: RCISES

COLUMBIA UNIVERSITY
DEPARTMENT OF PHYSIOLOOY
THE JOHN G. CURTIS LIBRARY

c

1l<^

y^

UNIVERSITY COLLEGE COURSE,

PEACTICAL BXEECISES

PHYSIOLOGY

J. BURDON SANDERSON, M.D., LL.D., F.R.S.

JODRELL PROFESSOR OF PHYSIOLOGY IN UNIVERSITY COLLEGE, LONDON.

WITH THE CO-OPERATION OF

F, J. M. PAOE, B.SC, F.C.S., W, NORTH, B.A., F.C.S., AND AUG. WALLER, M.D.

PHILADELPHIA
P. BLAKISTON, SON & Co., 1012 WALNUT STREET

1882

9?44

PREFACE.

The following- exercises are intended to serve as a guide to
the Practical Courses which are given in the Physiological
Department of this College. Part III. comprises the Chemi-
cal Exercises relating to Food Stuffs and Animal Liquids
which are performed by every Student in the Class Room, in
the ordinary course of Practical Physiology. These Exer-
cises were, for the most part, originally arranged by Mr.
Page. They have been used by many hundreds of Students
and have been found to work well. Part IV. contains direc-
tions for the more detailed practical study of the same subjects.
In the preparation of these, I have had the assistance of Mr.
North who has used them in the Practical Instructions which
he has given here. The exercises in Part I, relate to the
Physiology of Muscle and Nerve. In selecting them great
care has been taken to include nothing which cannot be suc-
cessfully carried out by the Student. Many of the exercises
have been contrived by Dr. Augustus Waller, who has de-

U PREFACE,

voted much time and thought to the simplification of methods.
The Demonstrations in Part IV. relate to various subjects.
These are separated from the rest of the course, on the ground
that they do not admit of being performed by each student
for himself.

UNIVERSITY COLLEGE COURSE

PRACTICAL EXERCISES IN PHYSIOLOGY.

Part I.

PRACTICAL EXERCISES RELATING TO THE PHY-
SIOLOGY OF MUSCLE AND NERVE.

1. Make electrodes as follows : — Prepare two straight, moder-
ately thick wires about four inches long. Taper each to a
blunt point at one end. Solder to the opposite end of each a
length of thin wire. Cover each with a thin layer of packing
wax. Prepare two three-inch lengths of glass tubing (which
should be thick walled and of narrow bore). Warm them,
and introduce the wires so that their points project half an
inch. Bind the tubes together for convenience of handling
and bare the wires by scraping the wax off near the point on
one side.

2. Put up a Daniell cell. The positive element is a well
amalgamated zinc rod immersed in ten per cent, sulphuric acid
contained in a porous cell ; the negative element is a copper
cylinder containing a solution of sulphate of copper. Put a
wire in each binding screw. The end of the wire attached
to the zinc (negative wire), is called the cathode; that at-
tached to the copper (positive wire), the anode.

B

2 PRACTICAL EXERCISES IN PHYSIOLOGY.

3. PitV'' a frog and prepare a sciatic Jierve without dividing it,
(See Hdb. p. 343). In the process the gastrocnemius should
not have twitched.

4. Connect the electrodes with a Daniell cell, interposing a key in
the circuit. Contraction follows make, or make and break. It
does not continue during* passage of current. The excita-
bility of the nerve is increased by division or injury.

5. Arrange cell and coil for single shocks, i.e., join the ends
of the battery wires to the two top screws of the du Bois' induc-
tion apparatus, in which the primary wire ends, interposing a
key by which the current is made and broken at will. Grad-
ually sliding the secondary towards the primary coil, observe
that the break shock is first responded to, then the make.
Note the distance of secondary from primary coil at which
you first get contraction in each case.

6. Arrange cell and coil for repeated shocks (faradisation),
by bringing the battery wires to the two screws A and E, Fig. i .
The circuit now includes the vibrating hammer or automatic
interrupter. On closing the current, the hammer is drawn
down and causes a break; the current ceasing, the hammer
is released, and contact is restored by the spring. You thus
obtain a succession of make and break induction currents in
alternately opposite directions. Prepare the second sciatic
nerve, and observe that faradisation produces continuous
muscular contraction or tetanus, which may be due either
to the series of break excitations, or to the double series of
strong break excitations and weaker ones at make. Note the
distance at which you first get contract4on.

7. To obtain successive make and break excitations of
nearly equal intensity, arrange for single shocks as in 5.

* In future experiments it is assumed that pithing is performed as a mat-
ter of course.

METHODS OF EXCITATION.

X "T^

JFi^ff.l

But in addition, connect the two ends of the primary coil
(the top binding- screws), by an extra derivation or "short
circuiting" wire broken by a key. By closing this key, the
current in the primary coil is diminished by derivation, and in-
creased to the same amount when it is opened. The diminu-
tion and increase give rise to induction currents, of which,
the directions are opposed like those produced by make and
break. Both of them are cat. par. weaker than the make
induction current, and they are sensibly equal to each other in
their excitatory effects.

8. Helmholtz' Modification of the Induction Apparatus.—
When it is necessary in faradising that the excitatory effects
of the make and break shocks should be equal, the apparatus
is arranged as in fig-. 2. Connect the battery wires as in 6.
Bridge the interrupter by a wire extending- from B to C.
Raise the upper contact screw C out of reach, and bring- the

62

PRACTICAL EXERCISES IN PHYSIOLOGY.

(3^

lower (F) within reach of the spring". Here, as in the other
case, the current in the primary coil diminishes by deriva-
tion, when the descending hammer touches F; increases to
the same amount when it rises, but is never broken.

9. Use of the du Bois key. Before proceeding further, note
that a key may be used for throwing a current into or cutting-
it off from a nerve or other excitable structure in \.\\o ways,
viz., (i) in such a way that when it is closed the current is
made, when it is opened the current is broken ; or (2) so that
when the key is closed it acts as a bridge, by which so large
a proportion of the current is derived, that it in effect vanishes
in the part of the circuit beyond the bridge.

In using induction currents for excitation, always employ
the second method.

10. To Cut off the Make or Break Shock.— For this pur-
pose a key may be introduced into the secondary circuit, by

METHODS OF EXCITATION. 5

which it can be closed or opened at will during make or
break of the primary current; or this may be effected auto-
matically, by fixing- an ebonite rod made for the purpose to
the hammer, so as to prolong it for about an inch. The rod
carries at its end a platinum wire in the shape of an inverted
U. The two limbs of the q are of such length that they
dip into two pools of mercury, which are severally connected
with the ends of the secondary coil. Consequently, when the
hammer descends, the [\ bridges the two pools, so that the
secondary coil is short circuited. As at the moment of break
of primary circuit the hammer is down, the break shock is
thus cut off. If the pools, instead of being connected as
above, are interpolated in secondary circuit, the make shock
is cut off.

II. Physiological Proof of the Break Extra Current.—
The extra current is the current produced in a coil by the
inductive influence of contiguous turns on each other, when a
voltaic current commences or ceases through the coil ; its
direction is against that of the battery current at make, with
it at break. Establish connections, as in Fig. 3, placing the

b PRACTICAL EXERCISES IN PHYSIOLOGY.

electrodes on the tongue. Close the key, A, so that the
current is cut off from the coil. Observe that opening and
closing the current by the key, B, produces little or no appre-
ciable effect. Now open the key. A, so that the current
passes through the coil. Opening the key, B, gives rise to a
Strong effect which is due to the break extra current.

12. Introduce a second pair of electrodes into the current of
the battery and primary coil, arranged as in 5, and apply
them to the tip of the tongue. Observe the effect of making
and breaking the current, first with, and then without the
core. Similarly compare these effects with those of the in-
duced make and break currents in the secondary wire.

13. Unipolar Excitation.— Connect one electrode with the
secondary coil, and apply it to the nerve. If the preparation
is completely insulated, there should be no response to make
or break. If the insulation is destroyed by touching the pre-
paration, or otherwise, contraction occurs.

It is to avoid unipolar excitation that, as a rule, the induc-
tion circuit is directed to be thrown in and out by using the
key as a bridge (See 9). Unipolar excitation is more apt to
occur with the break shock than with the make, in conse-
quence of the greater intensity of the former. Consequently,
it is avoided by using Helmholtz' modification. Prove this by
experiment.

14. Make and record observations on the comparative
excitability of nerve and muscle as follows : — Note the ap-
proximation (in centimeters) of the secondary to the primary
coil which is required to obtain a response (i) to the make
induction shock ; (2) to the break ; (3) to faradisation, with
the nerve undivided. Then repeat the observation after
dividing the nerve. Compare these effects with those ob-
served when the electrodes are applied directly to the muscle.

METHODS OF EXCITATION. 7

15. The Bitter- Valli Law (H.'"* p. 334). Prepare a sciatic
nerve without severing it. Compare the excitability to fara-
disation of the different parts of the nerve, placing- the
electrodes (i) at the ischium; (2) close above the knee: In
about an hour repeat the experiment, using- the same nerve.
Then sever the nerve near its orig-in, and so repeat the
experiments. Note the results in each case. Finally, repeat
the observations, using- the nerve of the opposite limb.

16. The Rheochord.— Whenever very weak voltaic currents
are required, we use the Rheochord. The simplest, and most
convenient form is a long- wire of German silver, of about 20
Ohms resistance, which, for convenience of space, is wound
on glass pegs which are fixed at equal distances in two rows
at opposite ends of a well-varnished mahogany board. The
wire is then divided into as many equal lengths as there are
pegs. On the board, underneath the first length is a scale,
each division of which is y-^-„ of the whole length of the wire.
The wire ends in two blocks, A and B, each of which has two
binding screws. In use, the battery wires are connected with
these two blocks. One of them (the one from the graduated
end of the rheochord wire) also receives the wire of the second
electrode. The other electrode wire is brought to a sliding
block, by which contact can be established with the rheochord
wire at any distance from A. In this arrangement, the cur-
rent through the nerve, or other structure to which the elec-
trodes are applied, is proportionate to the length of ivire between
the slider and the block. This relation would not hold good
were it not that the resistance of the nerve is always very
great as compared with that of the wire.

17. Polarisation of Electrodes.— Place a pair of electrodes
under the sciatic nerve, and join them by a key, and satisfy

• In this and other similar references, H. stands for Hermann, and F. lor
Foster.

8 PRACTICAL EXERCISES IN PHYSIOLOGY.

yourself that opening- and shutting the key gives no contrac-
tion. Connect the two wires of a Daniell with each side of
the same key, which, therefore, bridges the current. Allow
the current to pass through the nerve for a few instants by
opening the key, remove the battery wires, leaving the elec-
trode wires attached to the open key. Close the key, the
muscle will contract, the electrodes having been polarised by
the previous current. Leave the key closed for a few instants;
open and again close, the muscle will not contract, polar-
isation of the electrodes having subsided during closure of
circuit. This gives a reason for using the key as a bridge
to cut off the constant current. If, in experiments on
the law of contraction (See § 32) it is used to make and
break the current, it will be noticed that at each successive
closure the contractions diminish as polarisation augments.

18. Unpolarisable Electrodes. (H, 287, F. 55), The form
in common use consists of (i) a smooth, amalgamated*' zinc rod
dipping into (2) a saturated solution of zinc sulphate, with which
the tissue is electrically continuous by (3) a plug of china clay
made into a paste with saline solution -75 per cent. Threads
may be used to connect the plug with the tissue. Such elec-
trodes are of high resistance. (For the method of testing
them with reference to their freedom from polarity, See I., 17).

19. Galvani's Experiment.— Connect two dissimilar metals,
e.g., zinc and copper wire; and apply their points to a nerve, or
one point to a nerve, the other to any part of the frog; con-
traction occurs at make, or if the preparation is very excit-
able, at make and break.

• To make amalgamating liquid for electrodes, dissolve with gentle heat
3 c.c. of mercury in a mixture of 50 c.c. nitric acid, and 150 c.c. of hydro-
chloric acid. Dilute this liquid for use with its own volume of hydrochloric
acid, and eight times as much water.

METHODS or EXCITATION. Q

20. The Contraction without Metals.— Prepare a nerve-
muscle preparation, choosing- a vigorous and lively frog.
Lay the nerve on the muscles of the other limb stripped of
its skin. The muscle of the preparation contracts because
different parts of the surface with which the nerve is suddenly
brought into contact are at different potentials.

21. The Secondary Twitch. (F. p. 59, H. 290). Prepare
the sciatic nerve of one limb of a vigorous frog, and prepare
a nerve-muscle preparation from the other limb. Strip the
skin off the first limb, and lay the nerve of the preparation on
the gastrocnemius. Apply various stimuli to the first nerve,
both muscles contract; the secondary muscle contracts be-
cause its nerve is stimulated by the sudden electrical changes
which accompany the contraction of the primary muscle.

22. The Paradoxical Contraction. (H. 343, 346). Dissect
out one of the two main divisions of the sciatic, and divide it
at its periphery. Galvanic excitation of the peripheral part
of the divided branch, gives rise to contraction of the muscles
supplied by the other branch. The second nerve is stimulated
by the electrotonic alteration of the first nerve.

23. Action of Curare. (F. p. 38). la ) Inject one drop of
curare solution (i per cent.), having- stopped the circulation of
one limb by a ligature from which the nerve is excluded. In
a few minutes, test muscle and nerve of both limbs. Both
react on the ligatured side ; on the other side, muscle reacts,
nerve does not.

(b.) Proceed as before, but use the ordinary dose of curare,
I to 2 drops of o* I per cent, solution, and wait longer. In-
ject I drop of strychnia solution, (-05 per cent.) Pinching-
or touching curarised limbs, which have lost motility, will
excite reflex motion in the protected limb. Curare does not
paralyse afferent nerves or spinal centres.

lO PRACTICAL EXERCISES IN PHYSIOLOGY.

For these two experiments the frog is killed by de-
stroying- the hemispheres: the spinal cord must be left
intact.

(c.) Make two preparations of muscles with nerves A and B.
Of A allow the muscle to soak in curare dissolved in ^ per cent,
saline solution, keeping the nerve moistened with saline. Of
B allow the nerve to soak in curare. In about 15 minutes,
test muscle and nerve of both preparations; both muscles are
excitable; the nerve of A is inexcitable, that of B is excitable.
Curare does not paralyse motor nerve or muscle, but makes a
block between nerve and muscle.

24. Mechanical Excitation ; Mechanical Tetanus.— It has
already been seen that section or other mechanical injury is
an excitant of nerve, evidenced by one or more muscular
twitches.'"'' Connect a Grove cell with the "Tetanomotor,"
introducing a key into the circuit. The wire from the zinc
terminal of the battery must be inserted in the binding screw
marked jE, that from the platinum in Z, in Fig. i. Adjust the
apparatus so that on closing the key the ivory hammer vibrates
so as to excite, without destroying, a nerve placed on the
ivory groove. The effect produced is identical with tetanus by
faradisation.

25. Experiments in which the Graphic Method is
used. — For these experiments are required, (ij a revolving
cylinder tightly covered with paper, and then smoked, of
which the rate of rotation is known ; (2) suitable means
for supporting a muscle, so that it may act upon a lever
which presses lightly against the moving surface of the
paper.

* The positive element is a well amalgamated zinc, immersed in ten per
cent, sulphuric acid contained in a porous cell ; the negative element is a
sheet of platinum immersed in strong nitric acid.

THE MYOGRAPH. II

By noting the time required for a given number of revolu-
tions of the cylinder, and accurately measuring its circum-
ference, the rate of motion of the surface which is to receive
the record, may be fairly determined. But for more accurate
purposes, we must make a simultaneous record of the oscilla-
tions of a tuning fork, or of an electro-magnetic time-marker,
introduced into a circuit in which a vibrating reed acts as an
electro magnetic interrupter.

When for the purposes of the experiment, it is desirable
that the horizontal motion of the recording surfaces should
be slow, i.e., less then two inches per second, an interrupting
clock regulated by a pendulum or a metronome, is substituted
for the vibrating reed in the circuit of the time-marker. This
is so constructed that it makes and breaks the circuit at in-
tervals of one or more seconds.

26. The Myograph.— A simple and useful myograph is con-
structed as follows : — An oblong block of wood, on which a
cork plate is glued, supports the preparation. At one end of
the block is a vertical stem or pillar, of which the height and
distance from the preparation can be varied. This pillar is
surmounted by two steel points, which face each other at a
distance of about three quarters of an inch. On these the
axis of a bell-crank lever rotates, of which the horizontal
arm is prolonged by a rod of wood or thin vulcanite, and
ends in a writing point. To the vertical arm, the tendon of
the muscle of which it is desired to record the contractions,
is attached.

27. The Graphic Record or curve of a Single Contraction,
(twitch). (F. p. 42, H. p. 270). (a.) First arrange the re-
cording apparatus. For the present purpose the cylinder
must revolve about once in two seconds. With the apparatus
commonly used, a trigger key is provided, which can be so

12 PRACTICAL EXERCISES IN PHYSIOLOGY.

adjusted that the cylinder opens it on arriving- at a certain
point in its revolution. See that this key is in order, particu-
larly that when it is closed the contact is perfect. Cover the
cylinder with g-lazed paper, and smoke it over a petroleum
lamp. Cut off the hind limbs of a frog- just killed by pithing",
and sever them from each other. Place one of them on the
cork plate, in such a position that the tibia is in a line with
the long- arm of the lever. Expose the tendon of the gastro-
cnemius and after severing- and freeing- it from surrounding-
parts, tie to it a bit of strong- ligature thread immediately in
front of the sesamoid cartilage. Then expose the lower end
of the femur, and thrust through it a strong needle-point, to
which a thin wire leading from one of the binding screws at
the side of the block has been soldered. This serves to fix
the femoral attachment of the gastrocnemius. The needle in
which the wire from the other binding- screw ends, is to be
thrust through the tendon close to the ligature. Lastly, at-
tach the thread to the short vertical arm of the lever, bring
the myograph (which has up to this time been on the table),
into position, connect its binding screws with the wires of the
secondary coil of the induction apparatus, which should be at
a considerable distance from the primary. The next step is
to ascertain to what distance it is necessary to approximate
the secondary coil, in order to obtain a full response to break
induction shocks. This having been determined, open the
key in the primary circuit, bring the end of the writing lever
into contact with the smoked surface, ascertain that the
trigger key is in position, close it and allow the cylinder
to revolve slowly until the pin presses against the trigger.
Tap the tendon lightly, and set the clockwork in unrestrained
motion. As soon as the fly has attained its full expansion,
momentarily close the key in the primary circuit.

INFLUENCE OF TEMPERATURE. 1 3

(i.J Influence of Temperature on the Form of the Curve.
The prolong-ation of the single contraction produced by cold,
may be observed by placing" a few bits of ice in contact with
the skin by which the muscle is covered. To study the in-
fluence of heat, remove the ice, and place on the limb an
India-rubber bag filled with warm water; you find that the
contraction is shortened. If the water is above 40' C, the
muscle may become rigid.

fc.) Influence of Veratrin.— Inject a drop of O'l per cent,
solution of veratrin into the lymph sac of a brainless frog.
After twenty minutes, destroy the spinal cord and inscribe
one or more muscle curves, and compare them with those
previously obtained.

28. Superposition of two single contraction curves.—
(F. p. 47, H. p. 274), For the purpose of observing this it is
very advantageous to substitute the "pendulum myograph"
for the revolving cylinder. The pendulum is provided with
two trigger keys of the same kind as that employed in the last
experiment, which can be so adjusted that they are opened
by the pendulum in its swing. According to the distance at
which they are placed, two circuits in which they are severally
introduced, are broken at any desired time after each other.
Connect two Daniells with two induction coils. Arrange the
two trigger keys of the myograph, each in one of the two
primary circuits. Connect the two secondary coils by one
terminal of each ; connect the remaining two poles with
electrodes to the muscle, which must be fixed on the myo-
graph plate in the same manner as in (2).

Make two experiments, one in which the second excitation
follows the first at an interval of time shorter than one-
hundredth of a second, the other in which the interval is pro-
longed to five-hundredths. In the second there is accumu-
lation of effect, but not in the first.

14 PRACTICAL EXERCISES IN PHYSIOLOGY.

29. Composition of Tetanus. — Use a revolving cylinder
which rotates once in about ten seconds. The arrangement
of the circuit must be as in 4. A key is required in the se-
condary circuit (See 13) and an interrupter of the following
kind into the primary, (a) Fix one end of a steel spring con-
nected with one wire in a clamp so that when it is made to
oscillate the other end dips by its bent down point into and
out of mercury contained in a cup to which the other wire is
attached. Set the secondary coil at such a distance that the
break shock is alone effectual. Make several experiments,
first with the spring of such length that the interruptions
occur three or four times in a second ; a second with seven
or eight interruptions per second, and so on until the spasms
which were in the first experiment distinct become completely
fused, (b) Substitute for the spring and mercury pool an
interrupter which can be rapidly worked by the finger used
as in striking a pianoforte key. Strike as frequently and reg-
ularly as possible — say seven times per second. If the muscle
is fresh fusion will be incomplete. Paradise it once or several
times and repeat the observation.

30. Influence of fatigue. (F p. 88). (a) Arrange an ex-
periment exactly as in 27, but instead of closing the pri-
mary circuit for a moment only (see last line of paragraph)
allow it to remain closed. A series of contractions are re-
corded. The height of the contractions at first increases,
afterwards diminishes; the interval of time between the
moment of excitation and that at which the lever attains its
greatest height gradually increases. For the sake of distinct-
ness record only one in ten of the curves, for which purpose
the adjusting screw must be used to withdraw the lever from
the paper during the intervening contractions, (b) Repeat
the same experiment using a recording surface of which the

ELECTROTONUS. 15

rate of motion is not more than a miilim. per second. In this
case each contraction is recorded. It is seen that during" the
observations the rate of diminution of effect is uniform, so
that the line connecting" the apices of the contraction curve is
straight.

31. Experiments relating to Electrotonus. (F. p. 40, H.
P- 337)- I" these experiments as in 5 b. the slowly revolving
cylinder must be used. The muscle and nerve must be pre-
pared as follows: — Strip off the skin, cut across the trunk
half way down the back. Remove the viscera and the wall of
the visceral cavity. Lay the preparation on the cork plate,
ventral surface downwards. Expose the sciatic nerve in the
hollow of the knee. Thrust one blade of the scissors between
nerve and femur with its back to the nerve and divide the
femur about a third of an inch from the knee. Free the nerve
from surrounding parts following it to its origin, carefully
severing its muscular branches. Cut away everything ex-
cepting the remainder of the vertical column to which the
nerve is still attached, and lastly fix the cut-off end of the
femur in the clamp of the myograph. In all cases in which
as in the study of electrotonus, the nerve must be separated
from the surrounding parts, it is necessary either to support
and enclose it during the period of observation in a covered
trough, or to place the myograph and preparation in a moist
chamber, of such construction that the lever on which the
muscle acts is not enclosed.

The muscle chamber (F. p. 72) contains two pairs of non-
polarisable electrodes, the wires from which are connected with
binding screws outside. To these the following form is given
for economy of space. Each wire is soldered to a zinc rod, one
end of which is carefully polished and amalgamated. Over
the amalgamated end is drawn a sheath (like the finger of a

l6 PRACTICAL EXERCISES IN PHYSIOLOGY.

glove) of wash leather steeped in zinc sulphate solution. This
is further enclosed in a wider sheath steeped in o"6 per cent,
solution of chloride of sodium, the outside of which is smeared
with kaolin paste made with the same solution. The nerve
is supported by a vulcanite trough which is supported hori-
zontally by the pillar of the myograph in such a position
as to receive the whole of the nerve conveniently when
the preparation is in position. The sides of the trough
have notches at intervals, through which thick ligature
threads pass underneath the nerve; the opposite ends of
each thread are tied together below. Before putting the
threads in their places they are well soaked in salt solution
and smeared with soft kaolin paste. The ends of the threads
serve to bring them into connection with the sheaths of the
zinc rods, which are supported by their wires in a suitable
position for the purpose. Non-polarisable electrodes of this
form can only be used in the moist chamber.

Use two Daniells for the "polarising" current. Connect
them with the middle binding screws of a Pohl's reverser,
from which two other wires proceed to the end blocks of the
Rheochord (I. i6). Connect block A of the Rheochord with
one of the electrodes// and the slider with the other. Con-
nect the secondary coil of the induction apparatus with the
electrodes xx . Either single induction shocks or faradisation
may be used to test the excitability of the nerve. If the latter,
arrange the induction apparatus as in I. 8.

(a) Find the minimum distance of coil at which contrac-
tion occurs in the absence of the polarising current, and then
remove the coil just beyond that minimum. Make the polar-
ising current descending. If you are examining by single
shocks, you see at each excitation strong single twitches ; if
by faradisation you see that the muscle enters into tetanus.

LAW OF CONTRACTION. 1 7

It has thus been shown that the excitability is increased in the
vicinity of the cathode during the passage of the continuous
current. (b) Push the coil a little within the minimum, so
as to obtain evident contractions, or tetanus, according to
your arrangement. Make the polarising current ascending.
If you continue to test by single shocks, you find that they no
longer cause contraction; if by faradisation, that the tetanus
is cut short. The excitability is therefore diminished in the
vicinity of the anode during the passage of the continuous
current, (c) Employ single shocks to examine the after
effects, viz., the state of excitability after the passage of the
continuous current. You find that the excitability is increased
after the passage of the continuous current in either direction,
32. Demonstration of the Law of Contraction. (F. p. 75,
H. p. 338). For this purpose the arrangements required are
the same as for experiments on electrotonus, with the excep-
tion that the induction apparatus, and the battery and elec-
trodes connected with it, are not wanted. The key in the
primary circuit must be a mercurial one, and in perfect order.
Having completed the connections, close and open this key at
intervals of two seconds, so as to make and break the primary
circuit, gradually increasing the distance of the slider of the
rheochord from the block until the muscle begins to contract
at make. This effect is usually observed sooner, i.e., with a
weaker current when its direction is from the muscle. At
once shift the slider to the second length. The muscle will
probably respond to both make and break, whether it is
directed from or towards the muscle. Now substitute for the
two Daniells half a dozen Groves, or Lechanches, and dis-
pense with the rheochord. Under these conditions contraction
occurs at make only when the current is towards the muscle,
at break only when it is towards the spinal cord.

c

l8 PRACTICAL EXERCISES IN PHYSIOLOGY.

33. Bitter's Tetanus. (F. p. 76, H. p. 339). Conduct a
continuous current of such streng"th as to g-ive the third stage,
throug-h a nerve for a short time, the direction of which
should be towards the spinal cord. On breaking the current,
the muscle enters into tetanus, which can be instantly ar-
rested by again closing the current, or by cutting the nerve
near the muscle, but is not aboHshed by cutting the nerve
midway between the electrodes. It is sometimes possible to
observe the same effect on opening a current directed towards
the muscle. If so, it can be abolished by severing the nerves
between the electrodes. It is therefore dependent on condi-
tions which have their seat at the anode.

34. Experiments to Demonstrate the Seat of the Physio-
logical Influence of Fatigue.— Arrange two nerve -muscle
preparations, A and B, loaded equally with 50 grms. each,
with the nerves on the same pair of electrodes, connected
with the secondary coil. Below this point, on the nerve B,
apply the electrodes (unpolarisable) of a single Daniell, pre-
ferably so that the current is descending. Faradise, and at
the same time make the continuous current ; the muscle A,
will enter into tetanus, B will remain quiescent, the stimulus
being blocked by the electrotonic zone. Continue faradisa-
tion until the tetanus of A has quite subsided, then break the
constant current ; B will forthwith enter into tetanus. Both
nerves have been equally stimulated, and are, therefore,
equally fatigued; the tetanus of B shows that the excit-
ability of its nerve was not exhausted, and consequently that
the apparent exhaustion of A was not nervous.

Connect a nerve-muscle preparation with two pairs of
electrodes, one pair to the nerve, the other to the muscle.
Connect their wires to the two sides of a switch, so that you
can rapidly transfer the current from one pair to the other.

PERIOD OF LATENT STIMULATION. IQ

Paradise the nerve until tetanus has quite subsided, then
transfer the current to the muscle. The apparently ex-
hausted muscle enters into tetanus. For both experiments
the slowly revolving- cylinder should be used.

35. Measurement of the Period of Latent Stimulation by
the Pendulum Myograph. (F. p. 43, H. p. 271). Prepara-
tion of the apparatus. Cover the glass plate smoothly with
paper, smoke its surface as before, and fix it to the pen-
dulum. Arrange the "detent" and the "catch" so that
the pendulum, when detached from the former, just catches
on the latter. Test the instrument by taking tracings with a
tuning-fork, vibrating lOO times a second, on the smoked
paper, when the pendulum is moving at several different
velocities (the velocity varying with the positions of the de-
tent and catch). Arrange the electrical apparatus for single
shocks, as in 4, including in the primary circuit one of the
keys of the myograph. Prepare the gastrocnemius as in 27.
Great care must be taken in fixing the femur immovably
to the cork plate, in attaching- the ligature (for which thin
wire may be advantageously substituted) to the tendon
and lever. See also that no part of the apparatus touches
the surface of the glass plate, as the pendulum swings, except-
ing- the writing point, and that the pressure of the point on
the plate is slightly greater towards the end than at the
beginning of the swing-. Bring back the pendulum to its
place and see that everything- is in order — the keys closed,
the lever in its position, the electrodes under the nerve, etc.
On liberating- the pendulum, a muscle curve is inscribed on
the smoked surface. Withdraw the lever from its writing-
position, bring- the pendulum back past the key, close the
latter, keeping it closed by firm pressure of the finger, allow
the pendulum to rest against it, bring the lever into the writ-

c 2

20 PRACTICAL EXERCISES IN PHYSIOLOGY.

ing- position, and make a mark on the surface, which indicates
the moment of excitation. Take three or four similar curves,
depressing- the table an equal distance after each observation
(I or ^ turn) by the handle. Remove the muscle lever, and
take a tracing v^ith a tuning--fork, vibrating- lOO times a
second, carefully arranging the style of the fork in the posi-
tion previously occupied by the writing end of the muscle
lever. Remove the paper, varnish and measure the tracings.
From the mean result of the measurements, the latent stimu-
lation may be computed.

36. Rate of Propagation in Nerve. (F. p. 45, H. p. 345).
(a J In the frog. — The arrangements are the same as for the last
experiment. The nerve must be placed in a vulcanite trough
similar to that employed in 31, with the exception that in-
stead of threads, two pairs of wires cross the floor of the
trough at a distance of about an inch from each other. The
nerve must be prepared with great care, as in I., 3, and must
be in contact with both pairs of wires. Connect each couple
of wires with the side binding screws of a Pohl's reverser
from which the cross wires have been removed, and the wires
of the secondary coil to the middle screws, so that by turning
over the bridge, the near and the distant portion of the nerve
can be excited alternately. Cover the trough with a flap of
muscle, taking care that the flap does not touch the nerve.
Make a series of observations, throwing over the bridge
between each. Then take a tuning-fork tracing, varnish,
measure the length of nerve between the two contacts, and
calculate therefrom the rate of propagation, fd) The pen-
dulum myog-raph may also be used to measure the rate of
propagation in human motor nerve, the observer experiment-
ing on himself. Arrange a Marey's tympanum, so that its
lever may write on the glass plate. Connect the tympanum

ELASTIC PROPERTIES OF MUSCLE. 21

by an elastic tube in which there must be a small aperture
for the escape of air, with a pair of toy bellows held between
the thumb and finger of the left hand. Arrang-e the primary
circuit as before. Connect one electrode of large area from
the secondary coil with any part of the body. Apply a small
metal disc covered with wash leather steeped in strong- solu-
tion of salt to the skin, first at the bend of the elbow, and for
a second observation, above the clavicle, arranging the lever
of the tympanum so that the two curves shall be close to-
gether. Then make a scries of similar observations in pairs,
taking care to allow the pendulum to draw a base line to each
curve. Measure on the base lino the distance between the
curves of each pair, rejecting all records in which the two are
not of the same amplitude. The results obtained by this rough
method are surprisingly constant. They show that the rate
of propagation in man much exceeds that observed in the
frog.

37. The Elastic Properties of Muscle.— (a J For experi-
ments on this subject, use the slowly revolving cylinder, and
a counterpoised writing lever, two or three feet in length.
Arrange the gastrocnemius as in 31, substituting a scale
pan for the weight. Attach the tendon and scale pan to the
lever at as short a distance as possible from the writing
point. Prepare four equal weights of from 20 to 25 grammes
each, and cautiously place them in succession on the scale
pan while the cylinder is revolving. Observe that the exten-
sion is greater for the first weight, less for the next, and so
on ; and that the increase of length after each addition of
weight is gradual. On removing the weight, the muscle
resumes nearly its original length, which, however, it never
completely attains, although it continues to shorten for some
time after it ceases to be acted upon by the weight.

22 PRACTICAL EXERCISES IN PHYSIOLOGY.

(b) To compare the extensibility of tlie muscle during- rest
and action, load it successively with two weights, say of 50
grammes and 10 grammes. Determine the height to which
the point of the lever rises in tetanus with the two weights in
succession, and compare the difference between the two, with
the extension of the untetanised muscle, when successively
loaded with the same weights.

II. — The Frog Heart.

1. Rhytlimical Motions.— In a curarized preparation of
which the hemispheres have been destroyed, expose the ster-
num, and cut across the episternal cartilage. Then sever the
sternum from its connections by a cut on either side, and turn
it down over the belly. The heart is seen still covered by the
pericardium. Note the condition of each of its cavities, and
the mode of its rhythmical action.

2. The Inhibitory Centre. (F. p. 170). For the purpose
of observing the effect of passing series of induction shocks
through the inhibitory centre of the heart, a fine ligature is
attached to the fraenum (the thread-like ligament which
stretches from the dorsal aspect of the ventricle towards
the lower part of the pericardium). By means of the liga-
ture, the heart is raised out of its place and turned upwards.
The inhibitory centre is recognized by the whitish, crescent-
shaped line, which marks the junction of the wall of the sinus
with that of the right auricle. Paradise this spot for a second,
or less, placing the points of the electrodes on the line, a
couple of millims. distant from each other. Observe the
mode and order in which the cavities of the heart resume
thpir rhvthmical action.

THE FROG HEART. 23

3. Destroy the spinal cord by pithing, and observe the
changes thereby produced in the state of the circulation, and
particularly in the mode of action of the heart.

4. The Cardiac Vagus of the Frog.— (a) Preliminary Dis-
section.— Expose the trunk of the vagus nerve as it escapes
from the cranium as follows : — Remove the integument so as
to bring into view the muscles of the back of the neck on one
side, avoiding injury to the cutaneous vessels. Then expose
the scapula, and sever with the scissors the cartilaginous
from the bony scapula; remove the former, dividing the
muscles attached to it, then expose the sterno-mastoid muscle
which connects the outer part of the petrous bone and the
posterior border of the cartilaginous ring of the membrana
tympani with the concave anterior border of the scapula.
Remove or draw aside the sterno-mastoid so as to expose the
slender muscles (petrohyoidei) which run from the petrous
bone to the posterior horn of the hyoid bone, embracing the
cavity of the pharynx. Parallel with these muscles, and in
close relation with them, are seen the carotid artery and
several nerves, of which the two nearest the cranium are the
glosso-pharyngeal, and the vagus.

(d) Expose the vagus in a pithed preparation. Expose the
heart, as in 1, and introduce a small test tube into the
guUett. Fix the preparation in such a position on a cork,
that the electrodes can be conveniently applied to the nerve,
at the same time that the motion of the heart can be ob-
served.

5. The Stannius' Heart.—Prepare a frog heart with frae-
num ligature as before. Then pass a thick ligature under the
bifurcation of the aorta between it and the venae cavae superi-
ores. Then, seizing the fraenum ligature with the forceps,
turn the heart up. Carefully observe the position of the

24 PRACTICAL EXERCISES IN PHYSIOLOGY.

" crescent," and loop the ends of the ligature so that when it
is tightened it may embrace the crescent. On tightening, the
heart will stop in diastole.

In the heart so prepared, sever the ligatured parts from the
rest of the preparation with sharp scissors. The auricles and
ventricles resume their normal rhythmical action.

Cut off in a preparation which has been so treated, the re-
mainder of the auricles and the bulb, leaving the ventricle
and auriculo- ventricular septum. The heart continues to
beat normally, or, if the beats cease, they are renewed by a
pinch, by an induction shock, or by bringing a hot wire into
the neighbourhood of the cut surface.

6. Localization of the Motor Centres. -In one of two such
preparations (called ventricle preparations) which beat rhyth-
mically, cut off the whole of the auriculo-ventricular furrow
with sharp scissors. The preparation so obtained (the ven-
tricle apex) does not contract spontaneously, but responds to a
single excitation, whether mechanical or electrical, by a single
contraction, the duration of which is dependent on the tempera-
ture. In the other preparation, divide the ventricle by two
parallel cuts into a middle and two lateral thirds. The middle
third includes the ventricular border of the inter-auricular
septum, the right lateral third contains the root of the bulb.
The middle third beats rhythmically, the lateral thirds re-
spond to excitations by single contractions, but do not beat of
themselves.

7. Action of Muscarin and Atropin.— In an entire heart
(a heart removed by severing the vessels, for which purpose
the organ should be lifted out of the pericardium by a liga-
ture tied to the frsenum), stop rhythmical action by applying
to it a drop of serum containing a trace of muscarin. Ob-
serve the relaxed and motionless condition of the ventricle.

THE FROG HEART. 2$

After a few minutes, apply (in serum) a drop of 0*2 per
cent, solution of atropin. Observe the gradual restoration
of rhythmical action in the atropinized heart. Observe that
faradisation of the Inhibitory centre is without effect.

8. Action of the Constant Current on the Contractile
Substance of the Heart.— For this purpose prepare elec-
trodes as directed in i. Fix a cork vertically on a sheet
of lead about an inch and a half square ; cover the top
of the cork with wax mass, the upper surface of which
should be somewhat concave. Place the support on a sheet
of wet filtering- paper and cover it with a beaker. Attach a
fine ligature to the fraenum, and remove the heart after sever-
ing the principal vessels. Collect some blood and dilute it
with as much as 0*75 per cent, salt solution, and place a few
drops of it on the wax surface.

Make a "ventricle-apex preparation," as directed in 6.
Having ascertained that it does not beat rhythmically of itself,
fix it in its place by the aid of fine glass pins and replace the
beaker.

Prepare and arrange two Grove's cells in circuit, interpose
a key and a pair of electrodes. Fix the electrodes, so that
their points are in contact with the apex and base respectively
of the preparation. The passage through the ventricle apex
of a voltaic current in the direction of its axis, produces rhyth-
mical action, which lasts as long as the current passes.

9. Study of the Ventricular Systole by the Graphic
Method.— Prepare a writing lever consisting of a glass rod
about -^\^ inch in thickness, and five inches long, having at
one end a knob of glass, and at the other a writing point.
This is thrust through a square bit of cork, which is then
pushed up to the knob. A fine steel needle passes through
the cork at right angles to the rod. The rod also bears.

26 PRACTICAL EXERCISES IN PHYSIOLOGY.

close to the needle, a vertical arm of cork, by means of which
it rests on the ventricle. The preparation lies on a metal
plate, which forms the upper end of a cylindrical brass box,
through which water, at any desired temperature, can be
passed. This plate is furnished with bearings in which the
steel axis of the lever works. The metal box is fixed to one
of the adjustable supports of the recording apparatus.

(a) The rhythmically contracting heart.

Expose the heart as before. Raise it from the pericardium
by a ligature attached to the severed fraenum, and cut through
the vessels. Place the heart on a plate, adding a few drops
of dilute serum, and arrange the lever so that the cork arm
rests on the ventricle, and the writing end inscribes its move-
ments on the blackened surface of the cylinder. The rate of
motion should be about 20 inches per minute.

Allow water at 12° C. to pass through the cylindrical box
and record the rhythmical contractions of the ventricle. Re-
peat the experiment, substituting water at 17° and at 22°, and
compare the tracings. •

(b) The curve of a single ventricular contraction.
Prepare finely pointed electrodes, as in I. i, arranging for

single induction shocks. Fix the electrodes to an' adjustable
support, so that they can be brought with precision into con-
tact with the preparation. Prepare a Stannius' heart and
arrange it for recording as in a. Adjust the electrodes,
taking care not to interfere with the lever. Place the secon-
dary coil at about 10 centimeters distance from the primary,
or nearer, if on trial it is found necessary to do so. Then
bring the point of the lever into contact with the blackened
paper, so as to write a base line or abscissa, and open the
key. The rate of motion of the recording surface should be
about 2\ inches per second.

SPINAL AND OTHER REFLEX CENTRES. ^']

In order to obtain series of tracings which can be con-
veniently compared, introduce into the primary circuit the
self-acting" key described in I., 28. In this way a number of
curves may bedrawn on the same abscissa, or on parallel
abscissae at convenient distances from each other. Having
practised one or other of these methods, proceed to make the
following observations : —

a. When a succession of ventricular curves are drawn at
temperatures varying from 12° to 18°, C, it is found that the
duration of the systole is increased by about o"'i for every
degree of temperature.

/3, When the ventricle is excited by single induction shocks,
following each other at about 10" intervals, each curve is
observed to exceed its predecessor in amplitude, the aug-
ments gradually diminishing from the beginning to the end
of the series.

y. In the muscular tissue of the heart, the period of latent
stimulation is much longer than in voluntary muscle. Its
duration is about o"*i5. To measure it, a vertical line must
be drawn on the recording surface, indicating the position of
the writing point at the moment that the trigger of the cylin-
der comes into contact with the lever of the self-acting key.
{See I., 28).

III. — Functions of the Spinal and other Reflex Centres
OF THE Frog.

!• (F- 537» H- 479)- The preparation to be used in the
following experiments is obtained by severing the spinal cord
immediately behind the medulla oblongata, and introducing,
by the opening made for this purpose, a wooden plug into
the cranial cavity, so as to destroy its contents. This having

28 PRACTICAL EXERCISES IN PHYSIOLOGY.

been done, it is placed on a sheet of moist filter- paper, rest-
ing- on its ventral surface with the hind limbs extended, and
covered with a bell jar. For a time it remains motionless,
but eventually assumes a position which differs but little from
that of a living- frog. Observe the differences.

2. Prepare half-a-dozen pieces of filter-paper, each an
eighth of an inch square, and some strong acetic acid.
Turn the preparation over, and after observing that the
natural position is not resumed, apply one of the squares,
after moistening it with acetic acid and drawing off excess
by touching with dry filter paper, to the inside of the right
thigh, and observe the result. Repeat the experiment, hold-
ing the right foot. Next, attach the preparation to a suitable
holder in such a way that the trunk may be steadily supported
and the limbs may hang freely, and apply the squares in suc-
cession to different parts of the surface, as e.g., to the skin on
either side of the tendo Achillis, or to either flank. Observe
in each case that the muscular response which results from ex-
citation of the same part of the surface of the body is always
the same.

3. Arrange a second preparation as last described, using a
holder so constructed that the limbs may be suspended at any
desired height above the table. Prepare several beakers of
water acidulated respectively with i, 2, 3, 4, and 5 per thou-
sand of sulphuric acid, and place some of each mixture in a
saucer. Beginning with the weakest of the acid liquids, bring
down the preparation with the rack and pinion, until the tip
of the longest toe is immersed. Repeat the experiment at
intervals of three minutes with the stronger liquids, in order,
carefully washing the foot after each excitation, by dipping it
into a beaker of water. Measure the time which intervenes
between the beginning of the excitation and the muscular re-
sponse in each case, with the aid of a metronome.

SENSATION AND PERCEPTION. 29

4. Observe carefully the attitude of a brainless frog when
left to itself, and its behaviour when placed on its back, on
an inclined surface, or in water, as well as when excited by
cutaneous stimuli, comparing the phenomena observed with
those which exhibit themselves in the spinal cord preparation.

5. Proceed as in i, substituting a preparation in which,
after destruction of the brain, a couple of drops of a 0"i per
cent, solution of sulphate of strychnia have been injected un-
der the skin of the back. Observe that instead of co-ordinate
muscular responses, cutaneous excitation produces, under the
influence of strychnia, paroxysms of convulsion, in which the
body and limbs assume a characteristic attitude.

IV. — Sensation and Perception.

I. Time Occupied in the Simplest Mental Processes.
(F. 594, H. 511J. To measure the time required for re-
sponding to a signal (reaction time or personal time), the
simplest plan is to arrange a battery circuit in such a
way that it is closed by the same act by which the ob-
server makes the signal, and that it is opened by the re-
sponse of the observed person. Whatever be the nature of
the signal, the requirements are: — (1) Two Grove's cells
arranged in circuit; (2) a break key, (a lever resembling
in shape a pianoforte key, which, when touched, breaks a
mercurial contact); (3) a du Bois' key; (4) an electro-
magnet with a light lever attached to its armature; (5) a
chronograph ; (6) a recording surface, of which the rate of
motion is not less than i foot per second. The battery, two
keys, electro-magnet, and chronograph, are arranged in cir-
cuit, and in such positions that the electro-magnet lever may
be in the neighbourhood of the observed person, and the

30 PRACTICAL EXERCISES IN PHYSIOLOGY.

du Bois' key, cylinder, and chronograph, in reach of the
observer. On closing- the circuit, the lever is drawn towards
the magnet and gives the signal. The signal may be an in-
duction shock through the tip of the tongue, (in which case
an induction coil must be in circuit in addition to the instru-
ments above mentioned), a touch on the hand given by the
lever, a sound, or a visible signal, such as a white disk, letter,
or number, suddenly brought into view^

2. Tactile and Muscular Sensation, (F. 529, 530, H.
463-469). In all the following experiments two persons must
take part, one of whom must vary the conditions without
the knowledge of the other, and note the results. In the
experiments relating to the sensations of pressure, locality,
and muscular exertion, the observed person must have his
eyes shut.

The appreciation of Temperature must be tested by im-
mersing the same surface successively in water of slightly
different temperatures. The smallest differences can be
detected when the temperatures of the liquids compared,
approximate 30° C.

To test the sensation of Pressure, the hand or other part
to be investigated must be entirely at rest, and supported on
a horizontal surface. The weights used must be moderate —
from a pound to four or five pounds ; in which case it will be
found that a difference between two weights of one-thirtieth
can be detected.

For testing the sensation of locality in any part of the sur-
face of the body, a pair of compasses is used, of which the
points are provided with cork sheaths, having smooth blunt
ends. The points being at first at such a distance that when
both touch the skin or mucous membrane of the tongue, they
are distinctly felt as two, they are gradually brought nearer

SENSATION AND PERCEPTION. 3I

until the two impressions blend into one. The smaller the
distance at which this happens, the finer is the sensation of
locality in the region investigated. Another method is that
of interrogation. The observer touches the skin, and asks
the observed person to designate the locality touched.

The sensation of muscular exertion is tested by experi-
ments, each of which consists in lifting in succession, two
weights, of which one is heavier than the other by a small
but perceptible difference; this difference is diminished at
each trial until it can no longer be appreciated. As it is
essential that sensation of pressure should be excluded, the
weight to be estimated must in each trial be enclosed in a
handkerchief, of which the corners must be held in the hand.

For the investigation of the sensation of taste and of the
limits of the gustatory region, four test liquids should be pre-
pared, viz., saturated solution of sulphate of quinine, 10 per
cent, solution of common salt, 3 per cent, solution of sugar,
and 0"i per cent, solution of citric acid. These liquids re-
present the four fundamental sensations, each of which may
be tested separately, or two alternately. In each experiment
a camel-hair pencil is dipped in the liquid, drained by touch-
ing it with filter paper, and applied for a moment to the sur-
face. To secure freedom from bias on the part of the observed
person, trials should be made in which tasteless liquids, or
liquids of different tastes are alternated in various orders,
care being taken to irrigate the surface between each trial
and the following one, with water.

The voltaic sensations of taste are experienced when two
zinc plates, which form the terminals of a Grove's element,
are applied respectively to the upper and under surface of
the tongue as far back as possible. As the effect differs ac-
cording to the direction of the current, a reversing key must
be introduced into the circuit.

Part II.
DEMONSTRATIONS.

I. — Mode of Measuring and Recording the Arterial

Pressure. — Use of Recording Apparatus.

The instrument used is called a kymograph. The arterial
cannula is a T-shaped tube of glass. By its stem, it is
connected with the manometer (a U-shaped glass tube con-
taining mercury). One branch of the T is drawn out and
bevelled so as to be easily introduced into the artery : to the
other is fitted a short piece of india-rubber tubing, guarded
by a steel clip. The stem of the cannula communicates with
the proximal arm of the manometer by an unyielding tube of
lead or gutta-percha. The proximal arm (that connected
with the cannula) also communicates by a long flexible tube
with a bottle containing solution of bicarbonate of sodium
under pressure. The manometer is fixed to the recording
apparatus, so that its oscillations are inscribed on the moving
surface. This is effected by means of a style carried by a
vulcanite rod, which floats on the surface of the mercury in
the distal (open) limb of the manometer. The recording
cylinder is driven by clockwork; it is either covered with
smoked glazed paper, or is fed by an endless roll of paper,
in which case, a sable pencil, charged with coloured ink, is
substituted for the style. The paper surface in either case
moves at a uniform rate of 20 inches per minute.

The artery used is the carotid of the rabbit. The distal
end of the prepared part of the vessel is ligatured. The

USE OF RECORDING APPARATUS. 33

proximal end is temporarily closed by a spring--clip. The
vessel having- been opened near the ligature, the cannula is
introduced and secured in its place by a second ligature, its
drawn-out end being directed towards the heart. This done,
the guttapercha tube of the manometer is connected with the
stem of the cannula, and the whole system filled with solution
of sodic bicarbonate under a pressure of about four inches of
mercury. On removing the clip on the artery, communica-
tion is established between the arterial system and the mano-
meter, which now records the variations of arterial pressure.
The tracing- exhibits larger (respiratory) undulations, on each
of which many smaller undulations Ccardiac pulsations) are
inscribed. It shows (i) that each contraction of the left
ventricle produces a momentary increase of arterial pres-
sure; (2) that the pressure increases after each inspiration,
and sinks in the interval ; (3) that during the rise of pres-
sure the pulsations are more frequent than during- the fall.
Excitation of the Cardiac end. of the divided Vagus by
faradisation, produces (if weak induction currents are used)
diminution of the frequency of the heart's pulsation and of the
arterial pressure. If strong-er currents are used, the heart is
arrested in diastole.

[N.B. — In each of the Demonstrations, I., II., and III., a
rabbit is used, which is rendered completely insensible by a
suitable anaesthetic, and is killed before recovery.]

II. — The Normal Respiratory Movements. Influence of
THE Vagus Nerve, and of its Centre. Apn(ea and
Dyspncea.

The motions of a metal plate which is kept in constant
contact with the posterior surface of the central tendon of the

34 PRACTICAL EXERCISES IN PHYSIOLOGY.

diaphragm of the rabbit, by the pressure of a spring, are
communicated by a long steel wire to the vertical arm of a
bell-crank lever. The horizontal arm of the lever is pro-
longed, and bears a style by which an enlarged record of
the respiratory motion of the diaphragm is inscribed on the
cylinder of the recording apparatus. The rate of movement
of the cylinder is the same as in the last demonstration.

The inspiratory contraction of the diaphragm is expressed
by the descent of the writing style, its relaxation by the
ascent, w^hich is at first rapid, but afterwards more gradual.

Apnoea. — When by excessive artificial respiration the circu-
lating blood becomes overcharged with oxygen, all respiratory
movement ceases. On discontinuing the injections of air,
the respirations after a time begin again ; at first they are
scarcely perceptible, but each exceeds its predecessor in
extent, until the normal is reached.

Dyspnoea.— When an atmosphere containing an inadequate
percentage of oxygen is respired, the opposite effect to that
described above is produced. The respirations become more
ample and more frequent, and the auxiliary muscles are
brought into action. No such effect is produced by an
atmosphere containing as much as ten per cent, of COj,
provided that the supply of oxygen is sufficient.

Excitation of the Superior Laryngeal Nerve.— Excitation
of the central end of the trunk of the superior laryngeal nerve
by faradisation, arrests the respiratory movements, the dia-
phragm becoming stationary in the position of expiration.
When extremely feeble currents are used, rhythmical move-
ments may continue at long intervals. Introduction of irritant
gases or vapours into the larynx produces similar effects.

Similar excitation of the central end of the divided vagus,
below the cricoid cartilage, produces effects which differ ac-

FUNCTIONS OF VASCULAR NERVES. 35

cording- to the strength of the induction currents employed.
When currents of moderate strength are used, the diaphragm
remains, during the excitation, in the position of inspiration,
the state of contraction being, however, usually interrupted
by momentary relaxations at short intervals.

III. — Functions of Vascular Nerves.

Constricting Nerves.— Division of the trunk of the sympa-
thetic opposite the cricoid cartilage, is followed by dilatation
of the central artery of the lobe of the ear on the same side,
and increase of vascularity. On comparing the temperature
of the congested lobe with that of the other side, it is found to
be two or three degrees higher. The pupil of the same side
is more contracted than the opposite one. Excitation of the
end next the superior ganglion produces constriction of the
central artery, and abolishes the congestion of the lobe.

Dilating Nerves.— Excitation of the central end of the
great auricular nerve (or of the posterior auricular) pro-
duces temporary vascular changes, which are identical with
those permanently produced by section of the sympathetic.

Depressor Nerve.— Excitation of the central end of the
divided depressor occasions general diminution of arterial
pressure (dependent on dilatation of the blood-vessels sup-
plied by the splanchnic nerves). If the vagi have been
previously divided, the diminution of pressure is not associ-
ated with any change in the frequency of the contractions of
the heart.

D2

36 PRACTICAL EXERCISES IN PHYSIOLOGY.

IV. — Movements of Circulation and Respiration in Man.

I. The Cardiograph and Sphygmograph.— fa^ Two re-
ceiving" tympana (cardiographs) are used. One is applied to
the seat of the cardiac impulse, the other to the carotid artery.
The two recording tympana with which these are severally
connected, inscribe the motion of the heart and that of the
artery respectively, on the same cylinder. The arterial ex-
pansion follows that of the heart at an interval of about
eig-ht-hundredths of a second. The duration of the ventri-
cular impulse is about three-tenths of a second.

(h) The sphygmograph having been adjusted so as to
record the radial pulse, a receiving tympanum on the carotid
is connected with a recording tympanum attached to the
frame of the sphygmograph, so that its lever writes on the
same surface as that of the sphygmograph. The interval of
time between the impulse of the carotid and that of the radial
is about the same as that between the carotid and the heart.

2. The Stethograph.— The changes of form of the thorax
in respiration are investigated by the measurement of the
diameters of the chest. The most important diameters are,
the antero-posterior (from upper end of sternum to third
dorsal spine, 150 millims., and from lower end of sternum
to eighth spine, 200 millims.) ; the transverse (at the eighth
rib, about 230 millims.). These measurements refer to an
adult male, as taken during the respiratory pause. The first
of these diameters increases about a millimeter, the second
about two millimeters, and the third about two and a half in
ordinary tranquil inspiration. These measurements, when
recorded by the stethograph, yield the " respiratory curve."

ELECTROMOTIVE PHENOMENA OF MUSCLE. 2>7

V. — Electromotive Phenomena of Muscle.

The most important instrument used is a Thomson's
Reflecting Galvanometer, of hig-h resistance, the terminals
of which are connected by insulated copper wires with
non-polarisable electrodes. These are in contact by their
clay plugs with the two surfaces to be compared.

To the needle of the galvanometer a light concave mirror
is attached, on which a beam of light falls and is focussed,
after reflection, on a divided screen. Thus the smallest
deflection of the needle (by which any electrical diff"er-
ence between the two contacts is indicated) can be exactly
measured. By means of a suitable shunt, either the whole,
a tenth, or other decimal fraction of any current flowing
through the circuit can be led through the galvanometer.

The scale-reading of the galvanometer is proportionate to
the current passing through it, but afl^ords no indication of
the difference of potential between the two surfaces compared.
For this purpose it is necessary to balance the current in the
galvanometer circuit due to the electromotive force of the
muscle, by an opposed current of which the electromotive
force is known. The instrument used for this purpose is
called a Compensator. {See fig. 4). Two blocks, A and B,
are connected by a wire. They are also in connection (i)
with the two poles of a Standard Battery (Z>) by wires, one
of which passes through a multiplier, and (2) by two other
wires, one of which passes through a rheostat, with the corre-
sponding poles of a battery of several cells. This must be of
such strength that when the resistance of the rheostat is made
as small as possible, the battery, D, is over compensated.
The resistance is then increased until the galvanometer (g)

38 PRACTICAL EXERCISES IN PHYSIOLOGY.

is at zero. Under these conditions the difference of potential
between A and B is equal to the electromotive force of the
standard cell. If, therefore, as shown in the diagram, a part of
the rheochord wire, Aa, included in the same circuit with the
galvanometer G, is of such length that the current due to the
electromotive force of the muscle, is exactly balanced, so that
there is no deflection, the difference of potential between the
two led off surfaces/^ and m, is to the e.m. f. of the standard
cell, as the distance, Aa, is to the whole length of the wire.

All of the electromotive properties of muscle may be de-
monstrated more advantageously and strikingly with the aid
of the capillary electrometer, than by the galvanometer, first
because it indicates differences of potential, irrespectively of
resistance, and secondly because transitory changes in the
electrical relations of two "led off" surfaces (such as those

ELECTROMOTIVE PHENOMENA OF MUSCLE. 39

which accompany the excitatory process in muscle) can be
observed by it.

1. Electromotive Phenomena of Musele.—The g^astro-
cnemius muscle of the frog" is used. One of the electrodes is
in contact with the convex surface of the muscle near its
upper end, the other with the expansion of the tendo Achillis.
In this arrangement the surface of the tendon is negative to
that of the muscle.

2. On exciting the muscle by faradising its nerve, a deflec-
tion takes place in such a direction as to indicate that the
electrical difference between the two surfaces is diminished.
After excitation the needle resumes its former position.

If the capillary electrometer is used, it is seen that the
mercury column oscillates during the period of diminution,
and that the number of oscillations per second corresponds to
the number of excitations to which the muscle is subjected in
the same time.

3. The electrode in contact with the tendinous expansion is
now brought near to its fellow, so that both contacts are now
muscular. They are nearly isoelectrical. On injuring the
lower of the two contacts mechanically, or by heat, it be-
comes at once strongly negative. On excitation of the nerve
by induced currents, the negativity diminishes as before.

4. Electromotive Phenomena of the Ventricle of the
Prog Heart.— A Stannius' heart preparation {See Part II., 5)
is "led off"" by contacts at its apex and base. If the
heart is uninjured, these surfaces will be found to be
nearly isoelectrical. On injuring- either surface it becomes
negative.

2. A normally contracting heart is led off by contacts
similarly situated. Each contraction is accompanied by a
deflection of the needle, indicating that the apex becomes first

40 PRACTICAL EXERCISES IN PHYSIOLOGY.

positive then negative. By injuring the apex, mechanically or
otherwise, the deflection becomes entirely positive.

3. A ventricle preparation (Part II., 6) is led off at apex and
cut surface. During contraction, the effect is similar, but the
negative deflection is much larger.

4. A ventricle apex preparation (which does not contract
spontaneously) is led off as above.' Its cut surface is at first
strongly negative to the apex. On excitation at the base by
a single induction shock, the ventricle contracts, its contrac-
tion being accompanied by a deflection indicating that the
apex becomes negative.

Part III.

ELEMENTARY EXERCISES IN CHEMICAL PHYSIO-
LOGY.

I. — Starch, Dextrin, Dextrose, Fat.

1 . Starch is insoluble in cold water.

2. It dissolves imperfectly in hot water; the liquid so
obtained is opalescent.

3. It g-ives a blue colour with iodine, which vanishes when
the liquid is heated, but returns on cooling, if the heating has
not been prolonged.

4. Dextrin is soluble in water.

5. The solution gives a red-brown colour with iodine,
which vanishes on heating.

6. Commercial Grape-sugar is a yellowish-brown, crumbly
substance, which is readily soluble in water. Its solution is
usually slightly coloured, and reduces alkaline solutions of
cupric hydrate.

7. The Copper test.— To a small quantity of ten per cent,
solution of cupric sulphate, add about 5 c.c. of the liquid to
be tested ; then solution of caustic potash drop by drop until
the solution is clear, and heat gradually. If dextrose is
present, the blue colour vanishes, and a yellow precipitate
appears of cuprous hydrate, or a red precipitate of cuprous
oxide.

8. Conversion of starch into reducing sugar. Boil about
50 c.c. of starch solution in a flask with a drop of 25 per
cent, sulphuric acid for five minutes. The liquid becomes
limpid. It contains in addition to dextrose much soluble
starch (Amidulin) .

42 PRACTICAL EXERCISES IN PHYSIOLOGY.

9. Fat- Lard is insoluble in water. By boiling with
potash it yields a solution of soap.

10. Decompose the solution by adding a few drops of
dilute sulphuric acid. On heating, a layer of fatty acid
collects on the surface.

1 1 . Microscopical Preparations.— Starch grains ; their dis-
integration by hot water; action of iodine on them. Crystal-
line forms of fatty acids.

II. — Milk, Flour, Bread.

1. Milk has (in London) usually an acid reaction, and a
specific gravity of from 1025 to 1030. After removal of the
cream, the specific gravity is higher.

2. Milk contains fat, sugar, and proteids.

a. Proteids.— Heat about 50 c.c. of milk to 40° C. in a flask.
Add a few drops of rennet- extract, keeping the milk at the
same temperature until a coagulation is formed.

b. Dilute 5 c.c. milk with eight or nine times as much
water, acidulate with a drop or two of acetic acid, and warm
as before. Strain off the coagulated casein through muslin.

c. When milk is filtered under pressure through a porous
disk, its casein, being particulate, remains behind. The
clear filtrate contains lactose (milk-sugar) and salts.

d. Sugar.— The strained liquid from a (whey) contains
lactose, which, like dextrose, reduces metallic oxides. Apply
the copper test (I., 7).

e. Fat.— The coagulated casein contains much fat (butter)
which can be extracted by ether. The ether extract, when
evaporated on paper, leaves a greasy stain.

3. Flour.— Wash about a desert-spoonful of sound flour in
a muslin bag.

ALBUMIN. 43

a. A milky liquid passes through containing- much starch
(I., 3) but no sugar.

b. After washing for some minutes, a sticky and tenacious
material remains on the muslin, which can be collected; this,
after further washing, forms an elastic mass (gluten) which
can be drawn out into threads, and on burning, gives off the
smell of burnt feathers characteristic of a proteid.

4. Bread. — Digest with warm water. The extract contains
starch (I., 3) and dextrose (I., 7). The residue consists prin-
cipally of starch and gluten.

III. — Albumin and its Acid and Alkaline Modifications.

r. Albumin.— White of egg (albumen) when diluted with
water, strained and filtered, yields a faintly opalescent liquid.
This liquid contains a proteid body, albumin, which diffuses
through an animal membrane with great difficulty (V., 3).

2. Such a liquid, containing 5 per cent, of albumen, is to be
used in the following experiments. It coagulates on heating
at about 70° C. if neutral.

3. To some of the liquid add a few drops of 0"i per cent,
solution of caustic potash, and warm gently for two or three
minutes. Boil. The liquid will no longer coagulate, the
albumin having been transformed into the alkaline modifica-
tion (alkali-albumin).

4. In a similar way treat another portion with a few drops
of very dilute sulphuric acid (O'l per cent.). Warm very
gently for not less than five minutes. On boiling no coagu-
lation occurs, the albumin having passed into its acid modifi-
cation (acid-albumin, syntonin.)

5. Cool some of the liquid obtained in 3. Colour it
with litmus solution, and add carefully very dilute acid.

44 PR.\CTICAL EXERCISES IN PHYSIOLOGY.

A precipitate falls on neutralization, which is soluble in
excess of acid.

6. Make a similar experiment with the liquid obtained in
4, substituting w^eak solution of potash for weak acid. A
similar precipitate occurs on neutralization, which is soluble
in excess.

7. Take three portions, of 5 c.c. each, of the original
liquid in three test-tubes, and colour them wath litmus.
Dilute the c i per cent, acid about 5 times, and add a drop
of it to one of the portions ; to another add a drop of potash
solution similarly diluted. Heat all three tubes gradually,
and note the temperature at which each coag^jlates.

8. Make alkali-albumin solution as in 3. Divide it into
two equal parts. To one add two or three drops of 10 per
cent, solution of sodic phosphate. Colour both with litmus,
and neutralize with w-eak acid. The portion without sodic
phosphate is precipitated. The other portion is not precipi-
tated until enough acid has been added to convert the sodic
phosphate present into acid sodic phosphate.

TV. — Chakacteristics of Proteids. — Peptic Digestion.

I . Tests for proteid bodies in solution.

a. To some of the albuminous liquid referred to in TIL, 2,
add strong nitric acid. The precipitate obtained turns yel-
low on boiling.

6. Cool the liquid in a and add strong ammonia. The pre-
cipitate assumes an orange tint (Xanthoprotein reaction).

c. To another portion add Millon's reagent. (Mercury is
dissolved in its own weight of strong nitric acid. The solution
so obtained is diluted with twice its volume of water. The

PROTEIDS. 45

decanted clear liquid is Millon's reagent). A precipitate is
formed which turns dull red on boiling.

d. To a third portion add solution of potassic ferrocyanide,
and a drop of acetic acid. A white precipitate appears.

e. Introduce a fourth portion of the liquid into a test-tube
containing" one drop of ten per cent, solution of cupric sul-
phate. On adding solution of potash, a violet colour is
obtained (compare v., 2, b').

2. Serum-globvilin.

a. Neutralize 5 c.c. of serum with a few drops of O'l per
cent, sulphuric acid. Dilute with about 75 c.c. of water, and
allow the precipitate to settle. The precipitate is insoluble
in water, but soluble in excess of acid.

b. Dilute 5 c.c. of serum with 75 c.c. of water, and pass
through it a stream of CO,. The liquid becomes turbid as in a.

c. Repeat b without dilution. No precipitate is formed.

d. Add to a saturated solution of sulphate of magnesium a
small quantity of serum. A copious precipitate is formed.

e. Pour over some fibrin contained in a watch-glass some
solution oL pero.xide of hydrogen. Bubbles of oxygen are
given off. If some tincture of guaicum be added, a blue
colour is developed. Gluten, potato peelings, and many
other substances develop a blue colour under the same con-
ditions.

3. Peptic Digestion.

a. Introduce some fibrin into a test-tube, and just cover it
with 0-2 per cent, solution of HCl. Allow it to stand for
forty-five minutes in a water-bath at from 35° to 3S' C. At
the end of this time the fibrin is swollen and transparent, but
has not dissolved.

b. Repeat a, using instead of hydrochloric acid, water to
which a drop of glycerine extract of gastric mucous mem-
brane has been added.

46 PRACTICAL EXERCISES IN PHYSIOLOGY.

The fibrin remains unaltered,

c. Repeat a, adding- a drop of the same extract to the acid
liquid. The fibrin dissolves gradually.

d. Colour with litmus the liquid obtained in c. Neutralize
carefully with weak solution of caustic potash (III., 6). The
acid albumin formed during the first stage of digestion is pre-
cipitated.

V. — Pancreatic Digestion. — Amylolytic Ferments. —
Glycogen.

I. Pancreatic Digestion.

a. Introduce 5 c.c. of one per cent, solution of sodium
carbonate, to which a couple of drops of glycerine extract
of pancreas have been added, into each of two test-tubes.
Boil one of them and allow it to cool. Add some boiled fibrin
to each, and place them both in the water-bath at 35° C.
Compare the changes produced with those observed in pep-
tic digestion (IV., 3, c).

b. Examine the liquid product of a pancreatic digestion,
previously prepared by allowing a finely divided ox pancreas
to digest itself in a i per cent, solution of sodium carbonate.
It is alkaline, and may have a characteristic and offensive
odour.

c. Boil some of this liquid after acidulating slightly. Albu-
min is coagulated.

d. Colour another portion with litmus, and neutralize
carefully (III., 5); alkali-albumin is precipitated.

e. In a liquid obtained by concentrating the product above
referred to, after having separated the greater part of the
proteids contained in it, test for Tyrosin by adding Millon's

GLYCOGEN. 47

reagent, and boiling'. The presence of Tyrosin is indicated
by the reddish colour assumed by the liquid.

f. From such liquids Leucin usually separates on concentra-
tion, and can be recognized under the microscope by its
crystalline form.

2. Peptones.— A solution obtained either by pancreatic or
peptic digestion can be used.

a. The solution yields no precipitate either by boiling or
by neutralization, but is precipitated by alcohol.

b. When concentrated and treated as in IV., i, e, it gives
a red instead of a violet colour.

The liquid product of the slow putrefaction of proteids
resembles in most respects that of pancreatic digestion. To
the latter, the presence of septic organisms is not essential.

c. Peptone, although more diffusible than other proteids,
does not diffuse through parchment paper.

3. Indiffusibility of Proteids.— Suspend a parchment paper
tube containing diluted blood, in a beaker of distilled water,
so that the two open ends are above the surface. The col-
ouring matter and proteids do not pass through the mem-
brane. The soluble salts pass through readily, and their
presence in the water can be recognized by the usual tests.

4. Amylolytic Ferments.— Prepare some starch solution
and ascertain that it contains no dextrose (I., 2 and 8). To
another portion add saliva, and place the tube containing the
mixture in a water-bath at from 35° to 38° C. After a short
time, the product will be found to contain dextrose.

5. Glycogen.

a. To an extract of liver (prepared by extracting the per-
fectly fresh organ with boiling water after washing) add a
solution of iodine in potassic iodide. The liquid assumes a
red colour identical with that yielded under similar circum-
stances by dextrine {^See I., 5).

48 PRACTICAL EXERCISES IN PHYSIOLOGY.

b. Repeat 4, substituting extract of liver for starch paste,
using the same precautions.

VI.— Bile.

1. Observe colour and reaction of ox bile. It is usually
brown. Neutralize and boil in a test-tube. Bile does not
contain albumin.

2. Add a few drops of bile to methylated spirit. Mucin
is precipitated.

3. Prepare a solution of syntonin (III., 4) by digesting-
albumin in water containing 0*2 per cent, of hydrochloric
acid. On the addition of a drop of bile, the mixture curdles
en masse. If a large quantity of bile be added, little or no
precipitate may be formed, the liquid being rendered alkaline.

4. Boil bile with three-times its bulk of strong hydrochloric
acid for ten minutes. The bile is decomposed into bile-resin
(cholic acid with colouring matter) and glycin and taurin, the
two last-mentioned substances remaining in solution.

5 . Pettenkofer's Test for Cholic acid.— Spread a drop of
bile in a thin film on a white porcelain capsule. Mix with a
drop of strong solution of cane-sugar. Add concentrated
sulphuric acid drop by drop, and, if necessary, warm. A
deep purplish-red colour appears.

6. Repeat the test with an alcoholic solution of bilin. The
same colour is produced.

7. Gmelin's Test for the colouring matter. Spread a
drop of bile in a thin film on a white porcelain capsule.
Allow a drop of strong nitric acid to fall into the middle of
the film and observe the effect. The drop becomes sur-
rounded by rings of green, blue, red^ and yellow, in the
order in which they have been named. Consequently, Jhe

URINE. 49

gfreen, which is first formed, is eventually farthest from the
drop of acid. If, instead of allowing- the liquid to remain
undisturbed, the acid be mixed with the bile, the liquid passes
through the same tints, in the same order.

8. Warm a little nitric acid in a test tube. Incline the tube
and pour bile down the side, so as to form a layer over the
acid. The colours appear as in 7, at the line of contact of the
two liquids.

9. Cholesterin. An ethereal extract of gall stones yields,
on evaporation, crystals of cholesterin, which, when dropped
into warm sulphuric acid, dissolve with a red colour. The resi-
due, insoluble in ether, consists of colouring matter and mucin.

VII.— Urine, (i)

1. Observe reaction and colour.

2. Determine the specific gravity either by weighing- or
with the urinometer. Observe the effect of temperature.

3. Compare fresh with stale urine as regards appearance,
smell, and reaction.

4. Sulphates. Add baric chloride after acidifying- with
hydrochloric acid. A white precipitate of baric sulphate ist
formed.

5. Chlorides. Add argentic nitrate after acidifying with
nitric acid. A white curdy precipitate of argentic chloride is
produced.

6. Phosphates. Add ammonic molybdate to urine which
has been mixed with half its volume of nitric acid. Boil. A
yellow crystalline precipitate falls.

7. Urea. To urine evaporated to one-third, add a drop of
nitric acid in a watch-glass. Glistening scales of urea nitrate
are abundantly formed in the liquid.

50 PRACTICAL EXERCISES IN PHYSIOLOGY.

8. Uric Acid. To a hundred c.c. of urine add 5 c.c. of
strong- hydrochloric acid. Allow the liquid to stand for forty-
eight hours. Dark red crystals of uric acid separate from the
liquid.

9. Uroolirome. Precipitate about 50 c.c. with lead acetate
and a drop of ammonia. Filter. The filtrate is colourless.
Scrape the precipitate from the filter paper into a capsule.
Mix with a few drops of strong sulphuric acid and add to
the pasty mass a little alcohol. Filter. The yellow filtrate,
on boiling- with excess of strong sulphuric acid, turns black.
Dilute the acid liquid with a large quantity of water. The
tiromelanine which separates in flocks is characterized by its
extreme solubility in ammonia. It can be precipitated from
its solution in ammonia by sulphuric acid.

10. Indigo. To 500 c.c. of urine add 250 c.c. of pure
hydrochloric acid. Allow the liquid to stand twenty-four
hours. A coppery scum floats on the surface. Filter.
Treat the filter first with ammonia to extract the urome-
lanine, secondly with cold alcohol, which acquires thereby
a red colour. On boiling the residue in alcohol, a blue
solution is obtained, which exhibits the absorption spectrum
of indigo-blue.

N.B. — In consequence of the large quantities which must be
used, this experiment cannot be carried out by each student.

VIII.— Urine. (2)

I . Quantitative determination of ITrea. Urea (CO N3H4)
when decomposed by suitable oxidizing- agents, yields CO3 H^O
and N. The most convenient reagent for effecting this de-
composition is an alkaline solution of sodic hypobromite.
The CO3 is absorbed by caustic soda. The nitrogen which
is disengaged is collected and measured in a suitable ap-

DUPRE S APPARATUS.

51

paratus. Every 37-3 c.c. of nitrogen, at ordinary pressure

and temperature, corresponds to o*i grm. of urea. The

hypobromite solution is prepared by adding" 25 c.c. of

bromine to 250 c.c. of a solution containing 100 grms. of

caustic soda.

Fig. 5.

The stopper and test-tube represented in the upper left hand of the figure takes the place
of the stopper, pipette and tube e j. The woodcut has been kindly lent by Dr. Dupr6.

Dupre's apparatus is used. Introduce 25 c.c. of hypo-

E 2

52 PRACTICAL EXERCISES IN PHYSIOLOGY.

bromite into the flask c. Measure off 5 c.c. of urine into the
test-tube, and close the flask with the caoutchouc stopper to
which the test-tube is attached. Open the pinch-cock d and
lower the measuring- tube a, until the surface of the water is
at the zero point of the graduation. Close the pinch-cock
and raise the measuring tube. If the apparatus be tight,
mix the urine gradually with hypobromite solution by inclin-
ing" the flask. Finally, tilt the flask so as to rinse out the
test-tube with the solution, and shake well for a few seconds.
Immerse the flask in a vessel containing water at the same
temperature as that in the jar. At the same time lower
the measuring tube. After two or three minutes, raise
the measuring tube again until the surfaces of the liquids
inside and out coincide. Read off the quantity of nitro-
gen which results from the decomposition of the 5 c.c. of
urine.

2. Quantitative determination of Phosphates. — When
solution of uranic nitrate or acetate is added in successive
quantities to a hot solution containing phosphates, previously
acidified with acetic acid, the whole of the uranium is precipi-
tated so long as any phosphate remains in solution as uranic
phosphate. As soon as an excess of uranic salt is present,
it can be detected by potassic ferrocyanide, which gives a
brown colour with uranic salts.

The standard uranic nitrate solution contains 35*5 grammes
in a litre. One c.c. corresponds to 0*005 gramme PzO^.

To 50 c.c. of urine add 5 c.c. of a solution containing 100
grammes of sodic acetate in 900 c.c. of water, to which lOO
c.c. of glacial acetic acid have been added. Heat the 55 c.c.
to So" C. Add the uranic nitrate solution, until a drop of the
mixture placed on a white porcelain slab gives a distinct
brown colour, with a drop of potassic ferrocyanide. Note

COLOURING MATTER OF THE BLOOD. 53

the quantity of solution used, and calculate therefrom the per-
centage of PjO^ in the urine.**

IX. — The Colouring Matter of the Blood.

1. Observe the solar spectrum, noting the positions of the
dark lines D, E, b and F, in relation to the colours. Com-
pare it with the spectrum of a gas flame, which shows no
dark lines.

2. Observe the spectrum of a flame coloured with sodic
chloride, noting the position of the bright yellow line.

3. Oxy-hsemoglobin.— Introduce defibrinated blood into a
test-tube, and observe its opacity when undiluted.

(a) Dilute by adding five to ten times its bulk of water.
Place the test-tube in front of the slit of the spectroscope,
direct it to a gas flame. The only light which passes through
is that of the red end of the spectrum.

(b) Add water until the green appears. Note the dark
space (absorption band) between the red and green.

{c) Dilute still further until the yellow-green light is dis-
tinguishable in the middle of the dark space, dividing the
single broad band into two.

(d) After a further addition of water, note that the band
nearest the D line is somewhat more sharply defined than the

* For details as to the hypobromite method, see Dupre's original paper in
the journal of the Chemical Society, 1877, vol. i. p. 534.

The method for the determination of PgOg is practised in this class as an
example of a volumetric process. For other methods relating to the urine
see Part IV. It is important to remember, that in order to obtain trustworthy
results, as scrupulous care must be taken in the measurement and collection
of the urine passed during the period of observation as in the analytical pro-
cedures.

54 PRACTICAL EXERCISES IN PHYSIOLOGV,

other. The spectrum is still shortened by the absorption of
its violet end.

(e) On diluting-, until the solution is almost colourless, two
faint bands are still visible.

(/) Map on the diagram the appearances observed in 3,
b and d.

4. Reduced Hsemoglobin. — To some blood diluted as in 3,
d, add a drop of solution of ammonic sulphide, and warm
g-ently. The colour becomes purplish. Place the tube in
front of the slit as before, and observe the change which has
occurred. A single absorption band, with ill-defined edges,
takes the place of the two bands previously observed. Map
its position on the diagram.

5. Alkaline Hsematin (Hsemocliroinogen). — Add to solu-
tion of blood, rather stronger than the last, a drop of solu-
tion of caifliStic potash. Warm gently ; the colour completely
changes. An absorption band appears to the left of the line
D, and much of the blue end of the spectrum is cut off.

6. Reduded Alkaline Hsematin. — To the solution obtained
in 5 add a drop or two of ammonic sulphide and warm
gently. Observe the change of colour. Dilute if necessary.
A strongly marked band is seen to the right side of the line
D, and a second less defined, which nearly coincides with the
line E.

7. co-haemoglobin.— Blood which has been acted upon by
carbonic oxide has a peculiar cherry-red colour. The two
absorption bands have nearly the same position as those of
Oxy-haemoglobin, but no change is produced when the liquid
is treated with reducing agents, as in 4.

Part IV.

LABORATORY EXERCISES IN CHEMICAL-
PHYSIOLOGY.

I. — Starch and its Derivatives.

1. Prepare potato starch by grating potatoes into water,
stir the mixture thoroughly, and allow it to stand. After
partial subsidence pour off the turbid liquid and set it aside.
Collect the white deposit of starch, mix it with a fresh quan-
tity of water, and again separate it by subsidence and de-
cantation.

2. Examine the product microscopically. Each granule
exhibits concentric markings, which become more distinct
after the partial action of solution of iodine. In the dark
field of the polarisation microscope, each shows a dark cross
on a bright area.

Digest starch in saliva at 45' — 50° C. for 3 hours. Examine
the insoluble residue of cellulose before and after treating
with solution of iodine.

3. Boil a portion of the deposit in water to obtain "starch
solution" and filter. Observe that the liquid is opalescent
and that when a beam of sunlight passes through it the beam
is luminous. When viewed transversely through a Nicol's
prism, held between the thumb and forefinger, and rotated,
the luminosity appears and disappears alternately. Compare
in this respect solution of sulphate of quinine. Starch sola-
tion is indiflfusible.

4. Grind some malt in a coffee mill, extract the meal with

56 PRACTICAL EXERCISES IN PHYSIOLOGY.

four or five times its volume of water at 30° C. Concentrate
the extract at the same temperature to one half.

5. Prepare some half per cent, solution of starch, to part
of it add one tenth of its volume of malt extract and place the
mixture in the w^arm chamber at 70° C. From time to time
test portions of the liquid with solution of iodine. As soon as
the liquid remains colourless after the addition of the iodine,
arrest further change by boiling. Concentrate some of the
product and pour it into an excess of alcohol. The precipi-
tate is achroodextrin, maltose remaining in solution. Filter,
evaporate the filtrate, re-dissolve the residue in water, and
prove the presence of a reducing sugar.

6. Treat the remainder of the half per cent, solution of
starch, with one-tenth of its volume of malt extract as before,
keeping it in the warm chamber for three hours. Divide the
product into two exactly equal parts ; in one determine the
reducing power by the volumetric method described below.
Add to the other, two per cent, of concentrated sulphuric
acid, boil for half an hour in a flask ; bring the liquid to its
original volume by the addition of water, and estimate the
reducing power by the same method.

7. Volumetric estimation of sugar by Fehling's method.
Dissolve 34-639 grm. of pure cupric sulphate in about 20O

c.c. of distilled water, also dissolve 173 grm. of pure double
tartrate of potassium and sodium (Rochelle Salt) in 500 to
600 c.c. of a solution of caustic soda of i"i2 sp. gr,, mix the
two solutions and dilute to a litre. 10 c.c. of the solution so
prepared are equivalent to "05 grm. sugar.

Dilute 10 c.c. of the above solution with 40 or 50 c.c. of
water, and heat to boiling in a white porcelain basin.
Now run in from a burette the sugar solution (previously
diluted so as not to contain more than one half per cent.

MILK. 57

of sugar) and continue to do so until the blue colour of
the copper solution disappears, i.e. until all the copper is re-
duced. Rea.d the burette; the quantity used contains 05
g-rm. of sug-ar, whence the total quantity and the amount of
dilution being known, the total quantity of sugar may be
easily calculated.

II.— Milk.

1. Determine the sp. gr. of a sample of new milk with the
hydrometer. Allow it to stand for a day and skim off the
cream and determine the sp. gr. of the skimmed milk. Add
water until it is reduced to the original sp. gr. Ascertain
what proportion of water is required for this purpose.

2. Curdling of Milk.

(a) By Rennet.— Warm 50 c.c. of milk to 45° C. and add
20 drops of commercial extract of rennet. Set it aside for ten
or twenty minutes; the coagulum resembles that of blood
in consistence; filter and preserve the whey.

{b) By Acid.— Dilute milk with eight volumes of water at
40° C, add a drop or two of acetic acid. Separate the curd
which forms by gentle shaking, and filter; keep the filtrate.

Take two portions of the curd and dissolve one in weak
caustic soda, and the other in lime water. Filter both from
undissolved particles, and add a little rennet to both filtrates.
The solution in lime water will coagulate, that in soda will
not,

(<r) Salt Solution Curd.— Treat milk with twice its volume
of a saturated solution of common salt with the addition of
powdered salt, and shake thoroughly. (This is best effected by
a machine ; it can, however, be done on a small scale by shak-
ing milk in a test tube with powdered salt). The milk coagu-
lates, and a clear filtrate free from butter and casein may be

58 PRACTICAL EXERCISES IN PHYSIOLOGY.

obtained. Test the filtrate for sugar and coagulable pro-
teids and estimate the former by the method already given.

Dissolve some of the curd in water and add rennet to the
solution, observe that it coagulates.

It is obvious from these experiments that there are impor-
tant differences between acid and rennet curd, and that cal-
cium salts are accessory to the action of rennet.

{d) Take the whey from two equal quantities of milk, one
prepared with rennet the other with acid. Determine the
sp. gr. of each, and the amount of proteids. Compare the
results carefully.

(e) Neutralize and boil acid or sweet whey. Filter and
concentrate. Crystals of milk sugar may be obtained.

(f) Shake milk which has stood for several weeks in the
warm chamber, and is strongly acid, with ether, and decant
the ethereal extract, allow the ether to evaporate, and ex-
haust the residue with water. The watery extract is strongly
acid, and has the characteristic sour smell of lactic acid.

(S) Volatile Acids of Butter.— Take about 5 grms. of
butter, and treat it with excess of strong solution of caustic
potash in alcohol, warming slightly. When the action is
complete drive off the alcohol in the water bath and exhaust
the residue with a little water, place the watery extract in a
retort with excess of phosphoric acid and distil gently. Col-
lect the distillate and observe its odour and reaction.

III. — Albumen of Egg and Serum.

I . Method of determining the temperature of coagulation.

"A glass beaker containing water is placed within a second
larger beaker also containing water, the two being separated
by a ring or cork. Into the water contained in the inner
beaker there is immersed a test tube, in which is fixed an

ALBUMEN. 59

accurately graduated thermometer, provided with a long nar-
row bulb. The solution of the proteid of which the tempera-
ture of coagulation is to be determined, is placed in the test
tube, the quantity being just sufficient to cover the thermo-
meter bulb. The whole apparatus is then gradually heated.
With the arrangement described the rise in temperature
takes place very slowly and equally throughout. Care being
taken to have as good illumination as possible (the best plan
being to place the apparatus between the operator and a well
lighted window) the experimenter notes the temperature at
which the liquid first shows signs of opalescence; he after-
wards notes again the temperature at which a distinct separa-
tion of flocculent matter occurs," — Gamgee's Physiol. Chem.,
vol. i., p. 15.

2. Dissolve albumen in twenty times its volume of ten per
cent, salt solution. Introduce some of the liquid into a loop
of dialysing tube (of parchment paper), previously tested by
filling it with water, and suspend the tube in a beaker 01
water. Test the external liquid from time to time for chlo-
rine, by the following process : —

3. Estimation of Chlorine in the Difiusate -.—Dissolve 3*44
grms. of pure fused silver nitrate in distilled water, and make
up the solution to a litre. Dissolve also i grm. of pure fused
sodium chloride in water, and dilute to one litre. Fill a bu-
rette with the silver solution, measure with a pipette, ten c.c.
of the sodium chloride solution, and place it in a beaker with
one drop of a solution of neutral potassic chromate. Now
run in the silver solution, cautiously stirring the mixture dur-
ing the process, until the red colour produced by each drop
of the silver solution, and which at first disappears on stirring,
becomes permanent. If the solutions have been accurately
prepared, ten c.c. of the silver solution corresponding to -o i
grm. sodium chloride will be required.

6o

PRACTICAL EXERCISES IN PHYSIOLOGY.

Construct a dialysing- cell by closing the ends of a length
of dialysing tube with india-rubber corks, one of which is
perforated for two glass tubes. Of the two tubes the longer
must reach to the lower cork and be bevelled at its end ; the
other must be cut off short. Through this arrangement send
a current of water from the tap. Fill a narrow cylindrical
vessel, not much larger than the tube, with serum, and plunge
the latter into it. Allow the water to flow for 12 hours and
observe the precipitation of the globulin.

4. Prepare Lieberkiihn's jelly, by adding strong potash solu.
tion drop by drop to undiluted white of egg, stirring vigorously;
cut up the jelly into small pieces, and throw it into a large
beaker kept full of water by an arrangement similar to that
described in 3. Keep the produce in water for further use.

5. Prepare the whites of two eggs, disintegrate them by
clipping with scissors, rinse with twenty parts of water by
volume. Introduce the mixture into a corked flask, which
must not be more than half full, and shake briskly. Strain
through muslin and filter.

6. Dilute serum of blood with water, so that the mixture
shall have as nearly as possible the same sp. gr. as the pre-
vious one (5).

Compare the properties of the two forms of albumin, ac-
cording to the following table : —

Serum albumin is coagulated be-
tween 60° C. and 75° C. It is not
readily precipitated by hydrochloric
acid. The precipitate is readily sol-
uble in excess.

The precipitate, by boiling, is
readily soluble in strong nitric
acid. Its solution is not coagu-
lated by ether.

Egg albumin is coagulated be-
tween 63" C. and 70° C. It is
readily precipitated by hydrochloric
acid. The precipitate is not read-
ily soluble in excess.

The precipitate, by boiling, is
soluble with difficulty in strong
nitric acid. Its solution is coag-
ulated by ether (if the solution be
not alkaline).

GLOBULIN. 6l

IV.— Globulin of Serum.

1. Paraglobulin or Pibrinoplastin.— When solid magne-
sium sulphate is added in large excess to serum, and the
mixture violently agitated for some time by mechanical means,
a dense precipitate is obtained (serum globulin). This body
is an example of a class of proteids which are distinguished
by being insoluble in water, soluble in weak solutions of neu-
tral salts, but insoluble in saturated ones,

2. Collect the liquid in which the globulin has been pre-
cipitated, as above described, on several filters. It will re-
quire many hours for filtration. Preserve the filtrate.

3. After the filtration is complete, wash the filters with as
little water as possible, this will rapidly dissolve the precipi-
tate. The apparent solubility is due to the presence of
magnesium sulphate.

4. Add excess of magnesium sulphate in powder to the
clear filtrate from (2). It becomes turbid when shaken for
some time.

5. Test the coagulation point of the liquid as before
directed.

The Older Methods of Separating Serum Globulin.

6. Panum's method :— Dilute serum with fifteen times its
bulk of water, and add four drops of twenty-five per cent,
acetic acid to every 160 c.c. of the mixture.

7. Alex. Schmidt's method :— Dilute serum with twenty
volumes of water, and pass a stream of COj through the
mixture, keeping it cool during the process.

8. Collect the precipitate on a filter and wash it with water
saturated with COj. It is insoluble in water that has not

62 PRACTICAL EXERCISES IN PHYSIOLOGY.

been boiled ; in boiled water it dissolves with great diffi-
culty.

N.B. — An additional precipitate may be obtained from the
filtrate, by treating- it with a trace of acetic acid. This was
formerly regarded as serum casein by Panum.

9. Add to the filtrate from 3, sodium sulphate in powder
in excess, a further precipitate is obtained. This is serum
albumin. The filtrate still contains a small quantity of a sub-
stance which gives the proteid reactions.

10. Properties of Pericardial Fluid :— Obtain pericardial
fluid from the horse slaughterers, remove the contents of the
pericardium with a clean glass syringe, choosing for the pur-
pose an emaciated animal, and taking the greatest possible
care to avoid admixture with blood. The liquid should be
clear, and of a pale amber colour.

1 1. If freshly collected, it either fails to coagulate, or coa-
gulates very tardily. Whether it coagulates or not, it yields
when treated as in I a large precipitate of globulin similar
to that of serum.

N.B.— If hydrocele fluid can be obtained, it may be used in-
stead of pericardial fluid.

12. Coagulation of Blood-Plasma ;— Prepare magnesium
sulphate plasma, by receiving two parts of horse's blood into
one part of a saturated solution of magnesium sulphate.
Great care should be taken to mix the liquids thoroughly im-
mediately. The mixture must stand in a cool place (prefer-
ably in ice) for two days, to settle. After subsidence, the
supernatant liquid must be carefully drawn off with a pipette.

13. Collect a quart of horse's blood in a wide-mouthed
stoppered bottle, and set it in ice immediately. After it has
been allowed to stand for two days, draw off the serum, and
strip off" the upper colourless stratum of the clot (buffy coat),
and place it in salt solution.

DIGESTION. 63

14. Dilute some of the magnesium sulphate plasma with
fifteen to twenty volumes of water. Place it in the warm
chamber at 3^° C. It coagulates.

15. Mix serum with pericardial fluid in about equal volumes,
and treat the mixture as in 14.

16. Add to pericardial fluid in a watch-glass, a fragment
of fresh buffy coat, and place it in the warm chamber. Coa-
gulation occurs.

V. — Digestion.

Natural Digestion.— The state of the digestive organs dur-
ing the process of digestion, can be best seen in a dog which
has been well fed with meat freed from masses of fat some
seven hours previously. The animal may be conveniently
killed by the injection of cyanide of potassium into the pleural
cavity.

I. Open the abdominal cavity freely, in order to examine
the condition of the lacteals and thoracic duct. Tie the stom-
ach off from the duodenum by two ligatures, dividing between
them ; do the same at the lower end of the ileum. Open the
stomach along the greater curvature, turn it inside out, and
collect the semi-solid pulp which fills it. Observe that there
is very little liquid. Dilute the contents to 250 c.c. with water
(if the dog is of moderate size), strain through muslin, and
subsequently filter, precipitate the acid liquid by boiling with
ferric acetate. (This may be prepared by precipitating a
solution of iron alum with lead acetate, allowing it to stand
overnight, and decanting the clear liquid). Filter and test
the filtrate for peptone, with alkaline solution of cupric hy-
drate. The solution should give no precipitate with potassic

64 PRACTICAL EXERCISES IN PHYSIOLOGY.

ferrocyanide and acetic acid. The same method may be ap-
plied to the contents of the intestine. Note the absence of
foul odour and (under the microscope) of bacteria.

2. Artificial Digestive Products. — Gastric Digestion.—
Place a portion of mucous membrane scraped off the stomach
in a beaker, with 02 per cent, hydrochloric acid at 38° C. in
the warm chamber. Observe its rapid disappearance, filter
the liquid, and test its digestive power by the method de-
scribed in 7.

3. Make a five per cent, solution by volume of white of egg
in 02 per cent, hydrochloric acid, add to it glycerine ex~
tract of stomach in the proportion of lO c.c. to the litre.
Leave it in the warm chamber for twelve hours; neutralize^
separate the neutralization precipitate by filtration, boil the
filtrate with ferric acetate as before directed, filter and test
the filtrate for peptone as before.

4. Pancreatic Digestion.— Test the activity of the gly-
cerine extract of pancreas by the method described in Part
III., IV.

5. Pound part of an ox's pancreas into a pulp, place it
in one per cent, solution of sodium carbonate in the warm
chamber, and allow the product to remain in the warm
chamber for several days. Strain some of it through muslin,
filter, boil the filtrate, and again filter. Boil the second fil-
trate with Millon's reagent.

6. Apply the same process to the extract of the intestinal
contents, and compare the results.

7. Griitzner's Method of Comparing the Digestive Powers
of Solutions.— This depends upon the fact that when fibrin
stained with carmine is subjected to the action of a digestive
ferment, as the fibrin is dissolved, the carmine is liberated
and colours the fluid, the amount so set free being estimated

DIGESTION. 65

by an artificial scale consisting- of ten solutions of carmine of
different strengths, equal quantities of the stained fibrin being-
mixed with equal volumes of the digestive liquid to be com-
pared.

The following materials must be prepared : —

I. Carmine solution for staining the fibrin; take i grm.
carmine and dissolve it in about i c.c. of strong ammonia,
add 400 c.c. of water, place it in a bottle loosely stoppered
until the smell of ammonia has become faint. 2. Stained,
fibrin; take perfectly fresh and clean fibrin, chop it very fine,
place it in carmine solution for twenty-four hours, strain off
the fluid, and wash in water until the washings are colourless,
squeeze out the excess of fluid, and keep in a stoppered bottle
with just enough ether to cover it. 3. Another solution of
carmine to serve as a standard of colour. This is made by
dissolving i grm. of carmine in ammonia as before, and dilute
with glycerine to 100 c.c.

Take 6 c.c. of solution 3, and dilute to 60 c.c. with water.
Charge ten test tubes with quantities of the mixture ranging-
from I to 10 cub. centims., and dilute each with water to
20 c.c. Cork the tubes and number them in order, according-
to the strength of the solution it contains.

With the aid of a measure to hold i c.c, which can be
made by closing one end of a piece of glass tubing-, and
grinding the open end, measure out two equal quantities of
stained fibrin, and place them in two test tubes A and B, each
of which contains 20 c.c. of 0-2 per cent, hydrochloric acid.
To A add a measured quantity of one digestive liquid, and to
B precisely the sam.e quantity of the liquid with which it is to
be compared. Place both in the warm chamber, and at re-
gular intervals, which must be carefully noted, compare each
(after shaking) with the colour scale.

r

66 PRACTICAL EXERCISES IN PHYSIOLOGY.

VI. — Urine.

1 . Preparation of Urea from Urine.— Evaporate the urine
to a small bulk. Add strong nitric acid (pure and free from
other oxides of nitrogen) in excess, keeping the mixture cool
during the addition of the acid. Pour off the excess of fluid
from the crystals of urea nitrate which are formed, set them
on a porous tile or perforated dish to drain, press as much
fluid from them as is possible. Add barium carbonate in
large excess, mixing thoroughly, dry on a water bath and ex-
tract with absolute alcohol ; filter, evapprate the filtrate to a
syrup on the water bath, and set aside to crystallize. The
product may be further purified by animal charcoal and re-
crystallization. In order to carry out this process properly,
a whole day is required.

2. A quicker method is the following. Take 20 c.c. of
urine. Add " baryta mixture," (two volumes of barium hy-
drate solution, and one volume barium nitrate solution, both
saturated in the cold), until no further precipitate is produced;
filter, evaporate to a thick syrup on the water bath, and ex-
tract with alcohol. Pour off the alcoholic extract, filter, and
again evaporate to dryness on the water bath, and take up
with water. Place a drop of the watery solution on each of
the two slides, add to one, strong nitric acid ; allow both to
crystallize, and examine under the microscope.

3. Biuret Reaction.— Heat urea to 150°- 160° C. Dissolve
the product in a little warm water and add a solution of cupric
sulphate in caustic potash; a characteristic violet tint is pro-
duced.

3. Dupre's Method. (See Part III., VIII.) — Urea, when
acted upon by alkaline hypobromites or red fuming nitric

URINE. 67

acid, splits up into carbonic anhydride, nitrogen, and water.
Tiiis decomposition forms the basis of Dupre's and other
methods of estimating- urea by measuring the amount of ni-
trogen produced in the reaction. In this method, only 92 per
cent, of the nitrogen of the urea is given off. Uric acid yields
less than half, the other nitrogenous constituents of the
urine, variable proportions of their nitrogen, Hippuric acid
is not decomposed. In the graduation of Duprd's apparatus
this error is taken into account.

5. Liebig's Method..— A solution of pure mercuric nitrate
having been prepared, of such a strength that 20 c.c. of it are
required for the precipitation of 10 c.c. of a 2 per cent, solu-
tion of urea, — of which, therefore, 10 c.c. correspond to 0"i
grm. of urea ; — add 20 c.c. of the baryta mixture (see 2) to
40 c.c. of urine, and filter. Measure 15 c.c. of the filtrate,
which represents 10 c.c. of urine. Place it in a beaker, and
run in the mercury solution, (each c.c. of which corresponds to
O'Oi grm. of urea), until on mixing a drop of the mixture with
a drop of a saturated solution of sodium carbonate on a white
tile, a pale lemon colour appears. Now read the burette,
and calculate as follows : — If 10 c.c. of urine contained 0.2
grm. of urea, it would require 20 c.c. of the mercury solution.
I.e., I c.c. for each grm. per litre. If the daily quantity of
urine were 1500 c.c. this would give a daily discharge of 30
grms. of urea.

This method approaches accuracy only when the quantity
of urea present is about 2 per cent. The chlorine in the urine
must also be estimated, and the quantity of urea indicated re-
duced by the subtraction of i gramme of urea for every 13
grms. of sodium chloride found.

6. Estimation of Chlorides by Liebig's Method.— The fol-
lowing solutions must be carefully prepared, a. A solution.

68 PRACTICAL EXERCISES IN PHYSIOLOGY.

of mercuric nitrate of such a streng^th that i c.c. shall corre-
spond to •01 g'rm. of sodium chloride. Dissolve 20 grms. of
pure mercury in boiling- nitric acid, until a drop of the acid
fluid does not cause a precipitate when added to a solution of
common salt ; then dilute to nearly a litre, b. A solution of
pure sodium chloride, 20 ^rms. to the litre, c. A solution
made by dissolving- 4 grms. of pure urea in lOO c.c. of diluted
water, d. A solution of sodium sulphate saturated in the
cold.

To prepare the standard mercuric nitrate solution, place
TO c.c. of the standard sodium chloride solution in a beaker,
together with 2 c.c. of the urea solution, and 5 c.c. of the
solution of sodium sulphate. Now allow the solution of mer-
curic nitrate to flow g-ently into the beaker from a burette
(with glass tap), stirring the mixture during the process. A
precipitate forms at first, and is redissolved on stirring. On
adding more of the mercurial solution, the fluid becomes opa-
lescent, but no permanent precipitate fcrms until the reaction
is complete. The strength of the mercurial solution having
been determined by several experiments, it is diluted so that
20 c.c. = 0-2 grm. sodium chloride = 10 c.c. of the standard
sodium chloride solution.

Precipitate the urine with baryta mixture as directed in 5.
Take 15 c.c. of the filtrate, make it very slightly acid with very
dilute nitric acid, and then run in the mercury solution cau-
tiously, uniil a permanent dense cloud, not aff"ected by vigor-
ous stirring, makes its appearance ; the number of c.c. used,
multiplied by O'Oi, gives the amount of chlorine as sodium
chloride contained in 10 c.c. of urine.

This process depends upon the fact that when mercuric
nitrate and alkaline chloride in solution are mixed, sodium
nitrate and mercuric chloride are formed. It is, therefore,

URINE. 69

not until all the chloride in the urine has been so decomposed,
that mercuric nitrate begins to combine with the urea pre-
sent, to form a permanent white precipitate. The necessity
for estimating- the chlorides when using Licbig's process for
the determination of the urea is obvious.

7. Estimate the sug-ar in a sample of diabetic urine by the
process already given in I. 7.

S. Uric Acid.— Boil serpent's excrement with a 10 percent,
solution of caustic soda. Dilute, allow the liquid to stand,
and decant the clear fluid and pour it into a large quantity of
water to which 10 per cent, by volume of hydrochloric acid
has been added. Allow it to stand twenty-four hours, wash
and preserve the crystals which are deposited.

9. Treat uric acid or a urate in a porcelain dish with dilute
nitric acid, and evaporate to dryness; after cooling allow a
drop of strong potash to run over the residue. A deep pur-
ple colour is produced.

10. Hippuric Acid— Take 200 c.c. of fresh cow's urine and
concentrate it on the water bath to iio c.c, add Hydrochloric
acid and set aside till the next day. Take the brown crys-
talline mass which forms, wash with cold water, press be-
tween folds of filtering paper, dissolve in as little boiling
water as possible, add a little pure animal charcoal and filter,
concentrate the filtrate and set aside to crystallize.

11. To obtain hippuric acid from human urine take (soon
after a good meal) 1 to i'5 grms. of benzoic acid in wafer
paper. Collect the urine of the rest of the day and following
morning. Evaporate to a syrup on the water bath, extract
with alcohol.

12. Dry some of the crystals obtained by the method above
given, II, between folds of filter paper and treat in a test
tube, observe that a red oil forms, and that ammonia and

70 PRACTICAL EXERCISES IN PHYSIOLOGY.

benzoic acid are given off as may be recog"nized by the smell.
13. Creatinin.— Precipitate 200 c.c. of urine with milk of
lime. Filter and evaporate to a syrup. Extract with large
excess of alcohol and filter. Add to the filtrate tv^o drops of
a perfectly neutral solution of zinc chloride. Set the liquid
aside in a dark, cool place for two or three days. Creatinin
zinc chloride crystallizes in rosettes, in vertical lines on the
sides of the vessel.

VII. — Urinary Deposit.

Urinary deposits may be divided into two classes, accord-
ing to the reaction of the urine in which they occur.

I. Deposit in acid urine. — («) The so-called lateritious
deposit which forms on cooling in clear healthy urine, of high
specific gravity, and acid reaction, consists chiefly of uric
acid and urate of sodium, coloured by the urinary pigments.
This deposit may be obtained from any acid urine by slightly
concentrating and allowing it to stand.

Allow the deposit to subside and decant the liquid from it.
Examine it under the microscope. If uric acid is present its
characters can readily be recognized. Separate the crystals
from the amorphous deposit of urates, by washing with water,
and decantation. Note that the urate is readily soluble in
warm water, especially if it contains a trace of alkali.

(d) Calcic oxalate is also deposited under certain conditions,
in acid urine. It may be readily recognized by the micro-
scope. To observe its characters add to warm urine a few
drops of solution of calcic chloride and not more ammonium
lOxalate than is sufficient to produce a very slight precipitate.

URINARY DEPOSIT. 7I

Allow the urine to stand over nig'ht. After the ingestion of
any considerable quantity of rhubarb, calcic oxalate is always
deposited.

2. Deposits in alkaline urine.— (a) Add to ordinary urine
a few drops of urine which is already ammoniacal, and allow
it to stand for a few days in a warm place.

The deposit is whitish and consists of amorphous basic
phosphates of calcium and magnesium, identical with the
amorphous precipitate which is thrown down when urine is
neutralized — along with which are seen crystals of phosphate
of ammonium and magnesium (triple phosphate) and minute
organisms, on the surface of the urine is a scum which con-
tains the same crystalline and organized forms.

(b) Filter ammoniacal urine (previously decanted from the
sediment) wash the filter and dry it, then dip it in alcoholic
solution of turmeric and dry. The paper if wetted with fresh
urine and exposed to a current of air becomes brown, owing
to the decomposition of the urea.

c. Add to urine ammonium chloride and traces of sodium
phosphate and magnesium sulphate. Urine thus treated
yields on addition of ammonia a precipitate which on stand-
ing becomes crystalline. Compare the crystals with those
obtained from ammoniacal urine.

d. Acidulate urine with acetic acid, then add calcium
chloride and sodium phos[)hate in relatively small quantities,
A precipitate is formed, which becomes crystalline, of neutral
calcic phosphate. This body is precipitated under similar
conditions in urine.

72 PRACTICAL EXERCISES IN PHYSIOLOGY.

VIII. — Muscle.

1. Creatin, from aqueous extract of muscle, or best, from
Liebig-'s extract. — Dilute the latter with fifty times its volume
of water and precipitate it with lead acetate, excess being
avoided. Filter, separate the lead by sulphuretted hydrogen
and again filter. Evaporate to small bulk and set aside for
a week to crystallize. Pour off the mother liquor, and add
three or four volumes of alcohol sp. gr. 0*982, filter, wash
with alcohol, dissolve all the crystals obtained in boiling
water, again filter and set aside to crystallize. Creatin crys-
tallizes in transparent, colourless, rhombic prisms, for which
those of common salt may be easily mistaken. They are
distinguished from them by appearing illuminated in the
dark field of the polarization microscope.

2. Sarkin or Hypoxanthin.— If the filtered alcoholic extract
of the mother liquor of creatin be evaporated nearly to dry-
ness on a water-bath, extracted with water and filtered, the
filtrate yields on the addition of ammonia and ammoniacal
solution of silver nitrate, a precipitate which consists of silver
compounds of hypoxanthin and xanthin. For the method of
obtaining these bodies, see Gamgee's Physiological Chemistry,

P- 329-

3. Acidification of Muscle.— (a) Test the reaction of a
fresh sectional surface of curarized muscle. For this pur-
pose use two strips of glazed litmus paper of different colours.
Place them edge to edge on a varnished board, and affix the
cut surface over the junction. The reaction is neutral or
feebly alkaline. Test it again half an hour later, it will be
found to be acid, notwithstanding that the muscle has been
kept in a moist atmosphere, or under mercury.

BILE. 73

(d) Repeat the experiment, keeping- the muscle at 45" C,
observe that it becomes acid much sooner.

(c) Repeat (a) after subjecting the muscle to prolonged
tetanus. This is accomplished by faradising the spinal cord
in a decapitated frog, after previous division of the sciatic
on one side, and comparing the reaction of the cut surface of
the muscle which has been tetanized with that of the other.

4. The production of carbonic acid. That this process is
dependent upon contraction may be shown by arranging" two
preparations of the hind limbs of a frog in closed vessels,
over lime or baryta water. Of these, one is continuously
tetanized, the other is not, and air (previously deprived of
its CO2 by passage through strong potash) is aspirated slowly
through both. If the experiment be continued for some time,
it is seen that much more carbonate of lime or baryta is de-
posited in the vessel containing" the tetanized muscle than in
the other.

IX.— Bile.

1. Mucin.— Add to bile diluted with an equal volume of
water, excess of alcohol. Mucin is precipitated; filter, wash
the precipitate with dilute spirit, and dissolve in lime water
and again filter. Add excess of acetic acid to the filtrate.
Test the alkaline solution of mucin with Millon's reagent,
nitric acid, normal and basic lead acetate.

2. Preparation of bile crystals.— These consist of a mixture
of glycocholate and taurocholate of sodium, and are to be
prepared as follows : — evaporate the bile to a thick syrup,
and extract with strong" alcohol, decolorise the alcoholic
extract with animal charcoal, filter and evaporate off the

74 PRACTICAL EXERCISES IN PHYSIOLOGY.

alcohol on a water-bath, extract the residue with absolute
alcohol, pour the solution into excess of ether, and set aside
to crystallize in a well-stoppered bottle.

3. Taurin.— Boil dried or inspissated bile, or bile crystals,
for some time with strong hydrochloric acid in a flask fitted
with a long tube for condensing, filter, evaporate to dryness,
extract with absolute alcohol, dissolve the residue in water,
and set aside to crystallize.

4. Colouring matters.— (a) Take dog's bile, acidulate with
acetic acid, add excess of chloroform, and warm gently ;
remove the chloroform with a pipette, and evaporate on a
water-bath, the residue is crude bilirubin.

ib) Extract gall-stones with ether. Keep the ethereal
extract for the cholesterin it contains. Allow a few drops
to crystallize on a slide. Examine the crystals under the
microscope, and apply the iodine and sulphuric acid test.
Boil the residue in water acidulated with hydrochloric acid ;
pour off the bulk of the fluid, and add warm chloroform.
The solution on evaporation yields crude bilirubin. Test
the solubility of the product in water, ether, alcohol, and
chloroform. Examine the solution before the spectroscope,
and observe that it shows no absorption bands.

X. — Glycogen of the Liver.

I. Feed a rabbit on carrots, and, five or six hours after-
wards, kill it by opening the carotids. Open the chest and
abdomen quickly. Insert cannulse into the vena portse and
vena cava superior respectively, and pass a gentle stream of
water through the liver until it becomes uniformly pale. Now
cut it out quickly, mince it, and throw the pieces at once into

GLYCOGEN OF THE LIVER. 75

boiling- water acidulated with acetic acid. Filter the liquid
hot, evaporfite to small bulk, and add large excess of alcohol.
The glycogen is precipitated as a flocculent powder.

2. Take the livers of two or three oysters, mince finely, and
throw the pieces into boiling acidulated water, or treat as
in I.

3. Dissolve a small quantity of glycog'en prepared as
above, in water, add a few drops of saliva, place the mix-
ture in the warm chamber for five minutes, and test for
sugar.

Printed by H. K. Lewis, 136 Gower Street, London.

',''''

'^.