B313-716-500 
LABORATORY EXERCISES 
IN 
PHYSIOLOGY 
ARRANGED FOR THE USE OF MEDICAL STUDENTS 
BY 
WILLIAM S. CARTER, M. D., 
4 a * 
PROFESSOR OF PHYSIOLOGY IN THE UNIVERSITY OF TEXAS, 
GALVESTON, TEXAS. 
AUSTIN, TEXAS 
A. C. Baldwin & Sons, Printers 
1918 Copyright 1916 
by 
W. S. Carter Laboratory Exercises in Physiology 
Arranged for the Use of Students in the School of Medicine, 
University of Texas. 
By 
WILLIAM S. CARTER, M. D., 
Professor of Physiology. 
THE BLOOD. 
1. The Reaction of Blood: Put a drop of fresh defibrinated 
dog's blood on neutral glazed litmus paper and after 20 or 30 
seconds wash the blood off with distilled water. The litmus 
paper can be glazed by dipping it into a solution of neutral 
gelatin and allowing it to dry. 
A faint blue color shows that fresh blood is slightly alkaline. 
The same reaction is obtained with blood serum. If the serum 
is clear and free from hemoglobin it is not necessary to wash it 
off with water as the serum does not obscure the change of color 
with litmus. 
In the same way take the reaction of fresh human blood ob- 
tained from the finger by a sterile needle after wiping the skin 
with cotton wet with alcohol. Record the result of each ob- 
servation. 
2. The Potential Alkalinity of Blood Serum: Take 10 c. c. 
of clear serum free from hemoglobin in each of two small beakers 
or in test-tubes. Add a few drops of phenolphthalein to one and 
a few drops of methyl orange to the other. Observe that neither 
changes color. From the point of view of physical chemistry 4 
Laboratory Exercises in Physiology. 
the blood may be regarded as practically a neutral fluid, since 
the faint alkalinity shown with litmus is not enough to change 
the phenolphthalein. 
Now cautiously add from a graduated burette or pipette a 
NaOH sol. to the serum containing enolphthalein, and 
in the same way, titrate HC1 sol. into the serum containing 
the methyl orange. Observe and record in each case the exact 
amount used until a permanent red color is produced. 
Now take two portions of 10 c. c each of distilled water and 
add one of the indicators used above to each. Titrate in the 
same way and record the result. Contrast this with the amount 
used with blood serum. Explain the difference. 
Can the power of the blood to neutralize acids be determined 
by the ordinary methods of titration ? How does the alkali exist 
in the blood? Does it give to the blood a distinct absolute alka- 
linity physically or a potential alkalinity physiologically ? 
3. The Specific Gravity of Blood: Mix one part of chloro- 
form with three parts of benzol in a hydrometer cylinder and add 
a little of one or the other fluid until a mixture is obtained 
which after thorough mixing has a specific gravity of 1.050 or 
1.055 as determined by a hydrometer. 
After stirring some defibrinated blood in a beaker, draw a little 
up in a pipette with a narrow point. Allow a drop of the blood to 
escape into the fluid and observe whether it sinks to the bottom or 
rises to the top. If the blood is heavier, add choloform and mix 
thoroughly until the blood remains suspended in the fluid; if the 
blood is lighter, add benzol until the specific gravities are equal- 
ized and the drop of blood remains suspended in the mixture 
after it has been stirred up. Now take the specific gravity of 
the mixture with the hydrometer. This corresponds to the spe- 
cific gravity of the blood. 
Repeat the observation, using a drop of fresh human blood, 
and record the specific gravity. 
How would the condition of hydremia, or an excess of water 
in the blood, influence the specific gravity? 
4. The Coagulation Time: The blood usually clots in 3 to 
5 minutes after it is shed, and the time required for clotting 
is taken as an index of the coagulability of the blood. Vari- 6 
Laboratory Exercises in Physiology. 
ous coagulometers have been devised, but the results vary 
considerably with the method used. The most important factor 
is the temperature at which the blood is kept after it is re- 
moved from the body. 
The most convenient coagulometer consists of a series of small 
loops of uniform size in a platinum wire (Biffi-Brooks' method). 
These loops are filled with drops of blood and the glass rod 
holding the wire is gradually pushed through- a perforated stop- 
per, so that one loop is submerged in physiological salt solution 
every half minute until the blood remains clotted in the loop 
and does not mix with the solution. The fluid is kept at the 
bodily temperature, but there is no uniformity in the tempera- 
ture of the air in the flask or bottle over the physiological salt 
solution. This constitutes the greatest defect in this method 
and explains why considerable variations occur in repeated ob- 
servations on the same blood. 
Perhaps the most satisfactory coagulometer consists of a glass 
pipette made into a small piston syringe to hold a drop of blood 
submerged in water until it clots. 
The outer glass tube is drawn out to a pipette point about three 
inches long and having a lumen of less than 1 mm. at the end. 
Another tube of suitable size is drawn out with a bulbous end 
so that a piece of rubber tubing tied over it serves as a piston 
when it is moistened. 
A flask of water at 38°C is prepared with a stopper having 
two perforations and a thermometer in one of them. 
The skin of the finger is cleansed thoroughly and a large drop 
of blood obtained with a sterile needle, noting the exact time 
when the drop begins to form. 
As quickly as possible the blood is drawn up into the clean, 
dry pipette by cautiously withdrawing the piston so that all the 
blood is taken up avoiding any mixture with air bubbles. 
The pipette is then immersed in the water up to the level of 
the top of the blood and every half minute a very small drop 
of blood is expelled into the water by gently pushing the piston 
down. This is continued until the blood can not be expelled, or 
until a clot forms and can be seen floating in the wTater after 
some of the blood has been forced out of the pipette. Either 
may be taken as the end reaction when the fluidity of the blood 8 
Laboratory Exercises in Physiology. 
ceases and the time that elapsed from the drawing of the blood 
is the coagulation time. 
5. The Amount of Sodium Chloride in Solution Which Is 
Isotonic with the Erythrocytes: Two graduated centrifuge 
tubes of the same capacity are filled with exactly the same 
amount of defibrinated blood and centrifugalized until the cor- 
puscles sink to the lowest level. The exact volume of sedimented 
corpuscles is noted in each tube. 
Then the serum is pipetted off of one tube and replaced with 
0.9 per cent NaCl sol. The corpuscles are stirred up in both 
tubes and centrifugalized again until no further sedimentation 
occurs. The volume of corpuscles in each tube is again observed 
and recorded. 
The 0.9 per cent NaCl sol. is pipetted off and replaced with 
1.0 per cent NaCl. The corpuscles are stirred up in both tubes 
and centrifugalized again. The volume of corpuscles in each 
tube is again noted and compared with the previous readings. 
The 1.0 per cent NaCl is pipetted off and replaced with 0.8 
per cent NaCl sol. The corpuscles are stirred up in both tubes 
and centrifugalized as before. Observe and record the result. 
The observation may be repeated with 0.7 per cent NaCl sol. 
Compare the results with the different solutions of sodium 
chloride and state which ones are hypertonic, isotonic and hypo- 
tonic, respectively. 
Explain why the volume of the corpuscles is changed by hyper- 
tonic and hypotonic solutions, respectively. 
6. The Osmotic Resistance of Erythrocytes: Take a series 
of ten test-tubes and from a graduated burette or pippette run 
into the first 6 c. c. of 1 per cent NaCl sol.; into the second 5.8 
c. c.; into the third 5.6 c. c., and so on to the end of the series, 
always making a difference of 0.2 c. c. between the successive 
tubes. 
Then add distilled water in sufficient amount to make the solu- 
tion in each tube up to 10 c. c., i. e., starting with the first tube 
containing 6 c. c. of 1 per cent NaCl, add 4 c. c. distilled water; 
to the second 4.2 c. c.; to the third 4.4 c. c., etc. 
When the fluids are mixed they contain a series of solutions of 
sodium chloride decreasing in strength by 0.02 per cent in each 10 
Laboratory Exercises in Physiology. 
successive tube. The first tube contains 0.6 per cent and the last 
0.42 per cent NaCl. 
Put into each tube a drop of fresh blood, shake moderately to 
mix thoroughly and allow the tubes to stand frpm 15 to 30 min- 
utes. Observe the color of the clear fluid above the sedimented 
corpuscles and note the lowest percentage of NaCl in which the 
fluid is not tinged with red. What is the lowest percentage of 
sodium chloride which retains the hemoglobin in the corpuscles 
and prevents laking of the blood ? 
7. Spectroscopic Examination of Hemoglobin Compounds: 
Direct vision spectroscopes are used. When one looks at a 
bright part of the sky with a direct vision spectroscope, held so 
that the spectral colors extend horizontally with the red at the 
left, numerous vertical lines can be seen running through the 
spectrum (Fraunhofer4 s lines). These may be seen more dis-' 
tinctly by focussing the eye-piece and adjusting the amount of 
light allowed to enter the spectroscope by turning the milled 
ring at the prism end. The position of the line D in the orange 
is especially noted; also the line E in the green. The dark D 
line seen by sunlight is replaced by a bright line when the spec- 
troscope is directed toward a sodium flame and this serves to fix 
the position of this line. 
(a) Oxyhemoglobin. Some defibrinated blood is diluted 
with about ten volumes of distilled water and filtered. Some of 
the laked blood is then placed in an absorption cell or a test- 
tube and further diluted until two absorption bands can be seen. 
Observe that the darker one occurs in the yellow, to the right 
of the line D; that the one to the right is much broader but 
fainter and extends into the green. 
(&) Reduced hemoglobin. Some ammonium sulphide solu- 
tion is added to the solution of oxyhemoglobin and it is then 
heated gently to the temperature of the body. Observe that re- 
duced hemoglobin is formed showing a single, broad band, not 
sharply defined, which occurs between the position of the two 
bands in the oxyhemoglobin spectrum. 
Reduced hemoglobin may also be prepared by adding Stokes' 
reagent to a solution of oxyhemoglobin. Stokes' solution must 
be freshly prepared by dissolving two parts by weight of fer- 12 
Laboratory Exercises in Physiology. 
rous sulphate in a little water, adding three parts by weight of 
tartaric acid, and then adding ammonia until the reaction is 
alkaline. 
(c) .Carbonic Oxide Hemoglobin. Illuminating gas is al- 
lowed to bubble through a solution of oxyhemoglobin for a few 
minutes. The fluid changes to a bright cherry-red color due to 
the formation of carbon monoxide hemoglobin. 
When examined with the spectroscope two absortion bands 
are seen in the same position as those in the spectrum of oxy- 
hemoglobin. The bands are not identical as the band to the 
right (in the green) is sharper and not so broad as the one with 
oxyhemoglobin. The CO-hemoglobin is more stable than oxy- 
hemoglobin and does not yield reduced hemoglobin when am- 
monium sulphide is added. This serves to distinguish the two 
compounds. 
(cZ) Methemoglobin. A few drops of a potassium ferricy- 
anide solution are added to some blood in a test-tube and the 
mixture is heated gently. The color is changed to a brownish 
or chocolate color. It is then diluted with water and examined 
with the spectroscope. The spectrum of methemoglobin shows 
a well marked band in the red and two bands, which are less 
distinct, between the lines D and E. 
The addition of ammonium sulphide causes the band in the 
red to disappear and the spectrum of reduced hemoglobin is 
formed. This distinguishes the methemoglobin compound spec- 
troscopically from acid hematin as the spectrum of this com- 
pound resembles that of methemoglobin very closely. 
8. The Estimation of Hemoglobin: The normal amount of 
Hb. in the blood of man is 13 to 14 per cent by weight. The 
absolute amount is not stated in the quantitative determina- 
tions made for clinical purposes. The hemometers or hemo- 
globinometers used clinically indicate by colorometric methods 
the percentage of the normal amount, i. e., the normal hemo- 
globin content of the blood is taken as 100. The calibration 
of some of the hemoglobinometers is too high so that 90 per 
cent may be regarded as the normal for healthy adults. 
(a) Tallquist's Scale. Prick the finger and allow a drop of 
blood of moderate size to form. With a piece of white ab- 14 
Laboratory Exercises in Physiology. 
sorbent paper soak up the blood. After about a minute com- 
pare the color of the blood with the standard colors by passing 
the stain on the absorbent paper back of small openings in the 
color scale. The percentage is indicated by the shade which 
shows the same depth of color. 
This method has the advantage of simplicity. The result 
can be determined quickly but it is not as accurate as the 
other methods. The colors in the scale vary by 10 per cent. 
This method may detect the presence of anemia but can not 
be depended upon to determine with accuracy the degree of 
anemia. 
(b) Sdhli's Hemometer. The standard color tube is a 1 per 
cent solution of acid hematin which is a very stable com- 
pound and keeps for years without fading. The blood taken 
for examination is added to dilute HC1, thus forming acid 
hematin so that both tubes have the same color and may be 
viewed in any light. 
A tenth-normal sol. of HC1 is placed in the graduated tube 
up to the mark 10. The HC1 may be prepared with 
sufficient accuracy by diluting 15 c. c. of strong HC1 up to 
1 liter with distilled water, or by taking 1.5 c. c. of strong 
HC1 in 100 c. c. of distilled water. 
A drop of blood of moderate size is obtained from the 
finger and the special pipette is filled a little above the 20 
c. mm. mark. Any excess of blood is wiped from the end of 
the pipette and care should be taken to see that the lumen is 
filled exactly to the 20 c. mm. mark after the end of the 
pipette has been wiped off. The blood is then quickly1, ex- 
pelled into the dilute acid. The lumen of the pipette is washed 
free from blood by drawing the acid up and expelling it several 
times. 
Distilled water is now added gradually to the tube and 
mixed well with the contents until the depth of color is the 
same as that of the standard color tube. The percentage of 
the normal is expressed on the graduated tube since its ca- 
pacity is' 2 c. c. at the 100 mark and 20 c. mm. of blood were 
used. Record the result of your observation. 
(c) Gowers'Hemoglobinometer: Haldane's Modification. The 
standard color tube of the apparatus originally devised by 16 
Laboratory Exercises in Physiology. 
Gowers consists of glycerin jelly tinted with carmine to the 
standard color of normal blood diluted to 100 volumes with 
water. These color tubes fade with age and Haldane has 
made the important improvement of substituting a sealed tube 
filled with a one per cent solution of carbonic oxide hemo- 
globin. This keeps unchanged for years. 
Haldane's modification also requires the passage of illum- 
inating gas into the mixing tube to insure complete saturation 
of the Hb. with CO. 
The graduated tube is filled up to the 20 or 30 mark with 
distilled water. Blood is drawn up to the 20 c. mm. mark 
in the special pipette and blown into the water. Any blood 
adhering to the glass is washed out of the lumen of the pipette. 
A rubber tube attached to a gas pipe is then passed nearly to 
the level of the water and illuminating gas is allowed to pass 
for a few seconds. The end of the tube is then immediately 
closed with the finger and the tube is tipped back and forth 
at least a dozen times so that the liquid passes from one 
end to the other to saturate the Hb. with CO. While the tube 
is agitated in this way it should be held in a cloth or paper, 
otherwise it becomes heated from.the hand and will spurt 
when the finger is removed. 
Water is then added, drop by drop, and thoroughly mixed 
until the depth of color is the same as that in the standard 
color tube. The mark at the level of the fluid in the tube shows 
the percentage of Hb. in the blood. Record your observation 
and compare with the result by other methods 
(d) Fleischl's Ilemometer. The blood is drawn up by 
capillary attraction into a small capillary tube, containing 6.5 
c. mm. Care should be taken that only the end of the tube 
touches the drop of blood and that the tube is not stuck into 
it, as any excess on the outside can not be wiped off easily. 
When the tube is exactly filled, without any blood on the 
outside, it is washed in a few drops of distilled water placed 
in one side of the cell. The capillary tube, held by the small 
metal handle, is first agitated in the water and then washed 
out and thoroughly mixed by adding more distilled water from 
a pipette until the chamber is filled. The other side of the cell 
is filled with distilled water. The two halves of the cell are 18 
Laboratory Exercises in Physiology. 
then exactly filled, without any meniscus, and covered with a 
cover glass, without any mixing of the fluids, to make certain 
that the surface is exactly level in each compartment. 
Under the chamber containing the distilled water is placed 
a tinted glass wedge which can be moved back and forth by a 
thumb screw. This instrument can only be used in a dark 
room with yellow light, from a candle or oil lamp, reflected 
through the cell. The thumb screw is turned until the tints 
of the two sides are matched and the percentage is then read 
off on the scale. Several observations should be made and an 
average taken, working rapidly so as to avoid fatigue of the 
retina. Record the result. 
ENUMERATION OF CORPUSCLES. 
9. Volume Per Cent Determined by the Hematocrit: Prick 
the finger and wait for a large drop of blood to collect. Fill 
the hematocrit tiibe by sucking up the blood, taking care that 
air bubbles do not get into the tube. When the tube is com- 
pletely filled with blood, place the end of the index finger, 
greased with vaseline to prevent capillary attraction, over the 
free end of the glass tube and remove the rubber tube from the 
other end. 
Now place the glass tube in one arm of the centrifuge with 
the pointed end inward. See that the spring holds it securely 
and that rubber washers prevent the escape of the blood. 
Place another empty glass tube in the other arm of the hemat- 
ocrit to balance the instrument. 
Set the centrifuge at full speed for two minutes. Read 
the volume per cent of corpuscles sedimented at the distal end 
of the tube. Then centrifugalize for another minute and make 
a second reading. 
The sedimented corpuscles constitute about half the volume 
of blood, so that each volume per cent corresponds to 100,000 
corpuscles per cubic mm. approximately. Record the result of 
your own observation and compare it with the count. Caution: 
Success by this method depends upon working rapidly and 
accurately. Quite uniform results may be obtained if a uniform 
speed of the centrifuge is used. 20 
Laboratory Exercises in Physiology. 
For clinical purposes it is inconvenient to use the hema- 
tocrit at the bedside, so that it is necessary to dilute and fix 
the blood. The pipette of the hemocytometer for counting 
leucocytes is used for diluting 10 c. mm. of blood with an equal 
volume of Muller's fluid or a 2 p. c. sol. of potass, bichromate. 
The diluted blood can then be mixed in the bulb of the pipette 
and blown out into a watch crystal at any time; the hematocrit 
tube can then be filled and the corpuscles sedimented. The 
percentage must be multiplied by 2 on account of the dilu- 
tion of the blood. 
10. Thcma-Zeiss Hemacytometer. A. For Counting Ery- 
throcytes cr Red Corpuscles: Select thq pipette for diluting 
1 c. mm. in 100 c. mm. Draw the blood up to the 0.5 c. mm. 
mark, taking care that exactly this amount is used after wiping 
the point of the pipette oft'; without any delay draw Ilayem's 
fluid up to the 101 mark, making a dilution of 1 in 200; rotate 
the pipette by rolling it between the fingers or shaking it to 
mix the corpuscles uniformly. 
Blow a drop or two out of the pipette and then place a small 
drop in the center of the cell. Cover this with a special cover 
glass having a plane surface. 
Caution: The drop should not be large enough to overflow 
the gutter around the central cell or it will lift the cover glass 
up and increase the depth of the cell. If this occurs, wash the 
slide off and take another drop of proper size. There should 
not be any fluid between the cover glass and the surface on 
which it rests, but one should see a play of colors reflected, 
when the slide is held in a position to reflect the light, if the 
two surfaces of the glass are in close apposition. 
The depth of the cell is mm.; each side of the small 
squares measures j0 mm. The cubic content of each small 
square is XXtc- m- m- To determine the factor, 
this is multiplied by the degree of dilution of the blood, viz., 
-joo = so7A)0 The average number of corpuscles found in each 
small square represents soo'ooo °f the number in 0.5 c. mm. 
of blood. 
With the low power see that the corpuscles are evenly dis- 
tributed ; then focus the high power on the corpuscles in the 
squares and count the number in four large squares, prefer- 22 
Laboratory Exercises in Physiology. 
ably the four corner ones, if the corpuscles are evenly 'dis- 
tributed in them. Average the number for each small square 
and multiply by 800.000. Record your results. For very ac- 
curate results from 100 to 200 small squares should be counted 
from two separate drops. 
Clean the slide by washing with water; wash the pipette 
with water twice; then dry by drawing up alcohol, or alcohol 
and ether, and draw air through the tube, but do not blow 
through it. If a clot should form in the pipette, displace it 
with a horse hair and wash with dilute acetic acid, but never 
use a wire to displace a clot. 
11. B. To Count the Leucocytes: A larger quantity of 
blood is used and it is diluted with 1 per cent acetic acid 
which should be freshly prepared. The latter lakes or dis- 
solves the red corpuscles without changing the leucocytes, so 
that a dilution of 1 to 10 or 1 to 20 may be used. A weaker 
solution of acetic acid does not dissolve the erythrocytes com- 
pletely and leaves groups of "shadows" which cause irregular 
distribution of the leucocytes. 
Select the special pipette for making a 5 or 10 per cent dilu- 
tion. Draw the blood up to the mark 0.5 and then fill the pipette 
to the mark 11 with freshly prepared 1 per cent acetic acid, taking 
care not to get any air bubbles mixed with the fluid in the pipette. 
After mixing the blood thoroughly by rolling the pipette between 
the fingers let a few drops, escape, and then place a small drop on 
the central table of the cell as before. Take the same precautions 
to avoid an excess which would cause an overflow into the 
gutter about the central part of the cell. Apply the cover 
glass as before. Count the leucocytes in each large square 
and find the average. 
In this case the number of leucocytes in each large square is 
of the number in 1 c. mm. of the blood (|X| 
c. mm.X-yo^il-- suVa )• Multiply the average for each large 
square by 5000 and record the result. 
The total number of leucocytes may be counted in all of the 
16 large squares and the double lines which separate them. 
That number should be multiplied by 200 since the cross-ruled 
ruled field is 1 sq. mm., the depth mm., and the dilution 1:20. 
Accurate results require counts from two drops which may 
be made from the same dilution. 24 
Laboratory Exercises in Physiology. 
12. Differential Count of Leucocytes: In determining the 
proportions of the different varieties of leucocytes, a thin film 
of blood is dried and fixed on the slide. (The method of stain, 
ing these cells by Wright's stain will be given in the laboratory of 
clinical medicine.) At least 200 leucocytes should be counted. 
In all these counts the two classifications are more or less merged, 
since the protoplasmic granules may occur in the large mo- 
nonuclear and transitional cells. The following varieties are 
usually spoken of in differential counts: 
(a) Small Lymphocytes. These are small cells, usually 
about the size of a red blood cell. The nucleus is relatively 
large and stains deeply. There is little cytoplasm. These 
cells do not have protoplasmic granules. They seem to come 
from lymph glands and lymphoid tissues and have very lim- 
ited ameboid movement. They constitute from 20 to 30 per 
cent of the leucocytes of the blood in the adult. In the child 
they may be relatively much more abundant, amounting to as 
much as 40 or 50 per cent of the leucocytes at times. 
{b} The Large Mononuclear Leucocytes or Large Lympho- 
cytes. These have large nuclei and a large amount of protoplasm 
with or without granules. They constitute about 1 per cent of 
the leucocytes of normal blood. 
(c) Transitional Leucocytes These cells are usually consid- 
erably larger than erythrocytes; the protoplasm may contain 
granules having different staining properties; the nuclei are 
indented or lobulated and stain well. They constitute from 
1 to 3 per cent of leucocytes of normal blood. 
(d) Polymorphonuclear Neutrophites. These cells have poly- 
morphous nuclei with a large amount of cytoplasm. Fine gran- 
ules occur in the protoplasm which take neutral stains. These 
are the most abundant of the different varieties, constituting 
from 60 to 75 per cent of the leucocytes of the healthy adult. 
In children the proportion of these leucocytes may be as low 
as 40 per cent when there is a corresponding increase in the 
number of lymphocytes. 
(e) Eosinophiles. These cells may be mononuclear or poly- 
morphonuclear. There is an abundance of protoplasm, con- 
taining coarse granules which stain deeply with eosin, or other Laboratory Exercises in Physiology. 
26 
acid stains. These cells constitute from 1 to 3 per cent of the 
leucocytes. 
(/) Basophiles. In size and shape they resemble the poly- 
morphonuclear neutrophiles. The granules are usually larger 
and only stain with basic dyes. With other stains they appear 
as vacuoles. They are very scarce in normal blood, consti- 
tuting considerably less than 1 per cent. They stain well with 
thionin, methyl blue or dahlia. 
13. Phagocytosis: Tn order to show this property of leuco- 
cytes an emulsion of water color lampblack in physiological 
salt sol. is injected intravenously into a dog one-half hour be- 
for the examination of the blood. A drop of blood is obtained 
from the ear and both fresh and fixed preparations are ex- 
amined with the high power (oil immersion lens) of the 
microscope. 
Observe the black pigment granules in the protoplasm of 
the leucocytes. Observe that the polymorphonuclear leuco- 
cytes contain the greatest number of pigment granules, i. e., 
show the greatest phagocytic activity. These cells have rela- 
tively a large amount of protoplasm and are also most active 
in ameboid movements. 
14. Simple Anemia Produced by Bleeding and the Regen- 
eration of Blood: This experiment is performed to show (1) 
the changes in the blood from hemorrhage; (2) the accuracy 
of the methods used clinically in determining the degree of 
anemia, and (3) the regeneration of the blood after a consid- 
erable part (about one-fourth) of it has been removed ex- 
perimentally. 
A healthy dog is selected and the weight of blood in its 
body is calculated by taking 7.7 per cent of the body-weight. 
The volume of blood is then determined by dividing its weight 
by its specific gravity, viz., 1.055. 
The ear is shaved and the skin cleansed so that several 
drops of blood can be obtained by pricking the ear for the 
following determinations of the normal blood: 
(a) Erythrocytes per c. mm. 
(b) Leucocytes per c. mm. 
(c) Hemoglobin. 
(d) Coagulation time. 28 
Laboratory Exercises in Physiology. 
To avoid the possibility of error two observations should be 
made in each instance and the results recorded. 
The skin of the neck is shaved and disinfected with tincture 
of iodine and alcohol. The external jugular vein is exposed 
and a cannula tied in its distal end with all the precautions 
of asepsis. A definite quantity of the blood, amounting to 25 
per cent of the total blood of the body, is then withdrawn 
into a graduate and the wound closed to prevent infection. 
At the end of the bleeding, or immediately after it, the 
coagulation time and the specific gravity of the blood are 
again determined. These observations are repeated in 15 min- 
utes and again one or two hours after the bleeding. At the 
latter time the erythrocytes and leucocytes are counted and 
the percentage of hemoglobin is determined. 
All of these observations are repeated on each of the two 
days following the bleeding. The enumeration of erythrocytes 
and leucocytes and the determination of the hemoglobin are 
repeated at the end of one, two, three and four weeks after 
the bleeding. The determination of the coagulatiqn time and 
of the specific gravity need not be repeated after the return to 
the normal. 
Make a complete protocol of t^iis experiment, giving a tab- 
ular statement of the different observations in separate col- 
umns and also the percentage of the normal after each ob- 
servation on the erythrocytes and hemoglobin. Answer the 
following questions, basing your conclusions upon the results 
of this experiment: 
1. What is the effect of hemorrhage upon the coagulability 
of the blood? How long does this condition last? 
2. What is the effect of hemorrhage upon the specific grav- 
ity of the blood? How do you explain the alteration in the 
specific gravity and how long does it last? 
3. Is the diminution in the number of erythrocytes propor- 
tionate to the percentage of the blood removed by bleeding? 
Is the erythrocyte count a reliable index of the degree of 
anemia ? 
4. Is the reduction of the normal percentage of hemoglobin 
in direct proportion to the degree of anemia as determined 
by the removal of a definite amount of blood? 30 
Laboratory Exercises in Physiology. 
5. Does a simple anemia, produced experimentally by bleed- 
ing, alter the color index of the blood, i. e., the percentage 
of the hemoglobin divided by the percentage of the normal 
number of corpuscles? 
6. What alteration in the number of leucocytes follows 
hemorrhage and how long does it last? 
7. How long does it take the blood to regenerate and re 
turn to the normal, as shown by its corpuscular and hemoglobin 
content, after it has been impoverished by the removal of one- 
fourth of the total blood of the body? How soon is the 
water of the blood restored as shown by the hydremia and 
consequent decrease in specific gravity? 
15. The Transfusion of Blood: Successful transfusion re- 
quires (1) that the blood shall be transferred in such a man- 
ner that it does not clot while outside the body or produce 
intravascular clotting (thrombosis) in the recipient's vessels; 
(2) that there shall not be any deleterious interaction between 
the blood of the donor and that of the recipient. 
The danger of coagulation can be avoided by using certain 
precautions which retard or prevent coagulation. These can 
be demonstrated quite as well by taking a part of an animal's 
blood out of the body and returning it to the vessels of the 
same animal, as by transfusing from one animal to another. 
Direct transfusion consists in uniting the blood vessels in such 
a manner that blood may flow from one individual to another 
while it is kept in contact with the intima, or in interposing 
cannulas and a blood chamber coated throughout with a thin 
film of paraffin so that the foreign surface can not be wetted 
by the blood. The presence of a harmless, non-toxic anti- 
coagulant, like a sodium citrate solution, in the blood-chamber 
retards coagulation still further and makes it possible to trans- 
fuse two or three times longer than with paraffined apparatus 
alone. 
Indirect tranfusion consists in receiving blood into a 2.5 per 
cent of sodium citrate in the proportion of 10:1, so that 
the mixture contains 0.25 per cent sodium citrate. This will 
keep blood from clotting for days. In the amount used for 
transfusion it is non-toxic and does not alter the coagula- 
bility of the blood. The citrated blood may be kept for a 32 
Laboratory Exercises in Physiology. 
week or more in a sterile flask on ice and injected intra- 
venously by introducing a needle in the vein. The indirect 
method has all the advantages of simplicity. It does not re- 
quire any special operative skill or complicated apparatus. 
The greatest danger of transfusion is hemolysis or the solution 
of the erythrocytes. The plasma of the recipient's blood may 
hemolyze the donor's corpuscles, or the plasma of the trans- 
fused blood of the donor may cause extensive hemolysis of the 
recipient's corpuscles. 
Preliminary examinations should always be made by mix- 
ing the blood of the prospective donor with that of the re- 
cipient in different proportions to determine the compatibility 
of the two bloods before transfusion is practised. The danger 
of hemolysis can be avoided in that way since it does not occur 
in vivo if it is absent in vitro. The method of making such ex- 
aminations of the blood will be given later. 
While the blood of one dog may be incompatible with that 
of another, just as isohemolysins occur in the blood of man, 
for demonstration purposes a small amount of clear, citrated 
dog plasma may be injected into the vein of a rabbit. Ex- 
tensive hemolysis occurs in a few minutes and the animal dies 
from cytolytic changes if a sufficient amount is injected. The 
blood of the dog is markedly hemolytic for the rabbit. 
Observe and describe the symptoms in the rabbit. 
16. A. Hemolysis by Physical and Chemical Agents: 
1. Effect of Distilled Water. Place a small drop of defibri- 
nated blood is a small test-tube and add four or five drops of 
distilled water. Mix the contents of the tube well. Observe 
that after a short time the mixture becomes dark red in color 
and transparent owing to haemolysis of the red cells. Confirm 
this by examining a small drop of the mixture under the micro- 
scope. Hypotonic solutions act in the same way but more 
slowly. 
3. Effect of a Hypertonic Solution. Repeat this experi- 
ment but use physiological salt (0.85% NaCl) solution instead 
of water. Observe that the mixture in the tube remains turbid 
and the color does not change, showing that the red cells remain 
in suspension and do not go into solution. Observe that under 
the miscroscope the cells show their normal shape and size. 34 
Laboratory Exercises in Physiology. 
3. Effect of a Hypertonic Solution. Repeat above experi- 
ment using a hypertonic salt (3% NaCl) solution. Observe that 
the mixture has a brighter red color, but remains opaque. This 
is due to the fact that the red cells are crenated and reflect more 
light. Confirm this by the microscope. 
4. Effect of Chloroform-. Repeat the last experiment using 
an isotonic solution containing chloroform instead of the solu- 
tions mentioned above. Observe that hemolysis occurs. Chloro- 
form probably acts by dissolving the lecithin in the corpuscles. 
Record the result. 
5. Effect of Bile Salts. Repeat the last experiment, using 
a solution of bile salts in an isotonic solution. Observe that 
hemolysis occurs. Bile salts produce hemolysis in the same 
way as chloroform and ether. Record the result. 
B. Hemolysis by Natural and Speciffc Hemolysins. 
Specific Hemolysis. Two weeks before the observation on 
hemolysis is to be made, a rabbit is injected intravenously with 
5 c. c. of a 10 per cent suspension of sheep's corpuscles. This in- 
jection is repeated twice at intervals of three days. On the second 
day before the observation the rabbit is bled and the serum allowed 
to separate from the blood. Tn like manner the serum of a nor- 
mal rabbit is obtained and likewise that of a dog or cat. A 
sheep is also bled, the blood defibrinated and centrifugalized. 
The serum is removed and preserved. After washing the sheep's 
corpuscles three times with physiological salt solution, a 5 per 
cent suspension of the corpuscles in physiological salt (0.85 per 
cent NaCl) solution is made. 
On the day of the observation half of the serum from the in- 
jected rabbit (so-called "immune serum") is heated at 55°C for 
30 minutes, and half of the dog's serum is heated at 60°C for 
15 minutes. This destroys the "complement" but does not de- 
stroy the "amboceptor" as shown by one of the following ex- 
periments : 
We now have the following to work with: (1) Normal rabbit 
serum; (2) Immune rabbit serum; (3) Immune rabbit serum 
which has been heated to 55°C for 30 minutes; (4) Sheep's 
serum; (5) Washed sheep's corpuscles; (6) Normal dog's serum; 
(7) Dog's serum heated to 60°C for 15 minutes. 36 
Laboratory Exercises in Physiology. 
By use of the miseroscope and test tubes make the following 
observations: 
1. Agglutination. Mix equal parts of normal rabbit's serum 
and washed sheep's corpuscles in a small test tube. After stand- 
ing a few minutes examine a small drop under the microscope or 
place a small drop of each of these fluids on a slide and drop on 
a cover glass. Observe whether clumping or agglutination 
occurs and record the result. 
2. Agglutination and Hemolysis. Mix in another tube one 
part of "immune rabbit serum" with three parts of washed 
sheep corpuscles. Note that the mixture is opaque at first and 
becomes transparent later due to complete hemolysis.' Examine 
a drop of the mixture under the microscope from time to time, 
or place separate drops of the two fluids on a slide and observe 
the hemolysis as the fluids mix under the cover-glass. If the 
effects are not observed at first, repeat the observations after the 
mixture has stood for some time and record the result of the 
experiment. 
3. Effect of Heating Immune Serum. Mix one part of im- 
mune serum heated fo 55°C with three parts of washed sheep's 
corpuscles. After the mixture has stood for some time, observe 
under the microscope as before and record the result. Agglutina- 
tion may occur without hemolysis. The complement is de- 
stroyed at 55°C., while agglutinins are usually only destroyed 
at 60°C. 
4. Normal Serum Added to Immune Serum Heated to 55°C. 
Mix equal parts of heated immune serum, washed sheep cor- 
puscles and fresh normal rabbit or guinea pig serum. After 
giving time for changes to occur, examine a drop under the mic- 
roscope and observe the transparency of the fluid in the tube. 
Record the result. Heating destroys the "complement" but not 
the "amboceptor" or immune body. Normal serum "reactivates" 
the heated serum. 
5. Natural Hemolysis. Mix normal dog's or cat's serum 
with washed sheep corpuscles and observe the hemolysis. Record 
the result. 
6. Dog's or Cat's Serum Heated to 60°C. Repeat the last 
experiment, using serum that has been heated to destroy the 
natural hemolysin, and record the result. 38 
Laboratory Exercises in Physiology. 
17. Testing Donor's Blood Preliminary to Transfusion. 
A. Rous and Turner's Method: Use the leucocyte pipette 
(1-10) rinsed with 10 per cent sodium citrate solution; then 
draw the citrate solution up to the mark 1; the pipette is then 
filled rapidly to the mark 110 with blood from a large drop from 
the finger or ear, and without pause the mixture is expelled into 
a small, narrow test tube. This gives citrated blood with ap- 
proximately 1 per cent of the citrate in the mixture. 
The pipettes hold only 0.25 c. c. of fluid and that amount of 
blood is easily obtained. There is no objection to the use of 
pressure in order to get a large drop of blood rapidly. If the 
flow of blood ceases before the pipette can be filled, it must be 
expelled at once into a small test tube to prevent clotting. This 
mixture with citrate may then be taken up again and the pipette 
completely filled from a new puncture. 
The pipette is rinsed with citrate solution and then with water 
after the blood has been collected from each individual. 
Mixing. The mixing is done in Wright's tubes (tubes drawn 
out with a capillary end). Two mixtures of citrated blood are 
made: (a) A mixture containing nine parts of the recipient's 
blood to one of the donor's; (b) a mixture of equal parts of the 
two. In case of emergency the former will suffice, since it will 
show the most dangerous possibility, viz., that the blood plasma 
of the recipient may destroy the corpuscles of the donor. 
The proportions used in mixing need only be approximate. 
The capillary part of Wright's tube is marked with a paraffined 
pencil, blood is drawn to the mark, and each column of blood 
separated by a small air bubble from the next one that is drawn 
up. To insure thorough mixing, each mixture is expelled on a 
slide and then drawn up in the pipette again. If the examination 
can not be made at once the tube may be sealed off. 
A small drop of the mixture is expelled on a slide from the 
capillary end of the tube and a large drop of physiological salt 
solution is superimposed on the citrated blood without mixing. 
A cover glass is put on, and the preparation examined under 
the microscope for agglutination. 
The salt solution dilutes the blood and gives a clear picture 
as it spreads out. 
When agglutination occurs it begins in two or three minutes 40 
Laboratory Exercises in Physiology. 
and increases for a time. Observations should be made every 
two or three minutes. If it does not occur in fifteen minutes, 
the bloods may be considered compatible. In that case, the cor- 
puscles are evenly distributed or in rouleaux as in an ordinary 
blood. 
When agglutination occurs, the characteristic clumping may 
be in small masses or sometimes in large ones which can be seen 
microscopically. When pressure is made on the eoverslip the 
agglutinated cells pull out in columns. 
Observe and record the presence or absence of agglutination 
in the first mixture (a) containing one part of the donor's blood 
to nine parts of the recipient's blood. This represents approxi- 
mately the proportion in which the two bloods will be mixed in 
the recipient's vessels after transfusion. Agglutination in this 
case indicates that the recipient's plasma agglutinates the donor's 
corpuscles. 
Record the result in the mixture (b) which contains equal 
parts of the two bloods. Agglutination in these proportions, 
especially if it did not occur in (a), indicates that the donor's 
plasma acts upon the recipient's corpuscles. This is less serious 
as the proportion in which the two bloods should be mixed in 
transfusion would in all probability not result in hemolysis. 
Agglutination may occur without hemolysis, but hemolysis is 
invariably preceded or accompanied by agglutination. It is, 
therefore, possible to avoid hemolysis by selecting a donor whose 
blood does not show any agglutination when mixed with the re- 
cipient's blood in the proportions given above. 
For purposes of study by the class, the citrated blood of man, 
the sheep and two different dogs will be used for mixing in 
different proportions to determine their compatibilities. 
B. Minot's Method of Determining the Compatibility of 
Different Bloods: Minot has modified and simplified the method 
of Rous and Turner given above. It has the advantage of 
diluting and mixing the bloods at once. This saves consid- 
erable time. An examination can be made by Minot's method 
in fifteen or twenty minutes which is about one-third of the 
time required for Rous and Turner's method. 
The results are practically as reliable and satisfactory, al- 42 
Laboratory Exercises in Physiology. 
though neither the dilution nor the mixing is done with the same 
accuracy. Minot's method is as follows: Three drops of a 1.5 
per cent solution of sodium citrate in 0.9 per cent NaCI sol. are 
placed in each of two watch crystals or small test-tubes. Tn 
the first one, nine drops of the recipient's blood are received 
directly into the citrate solution and mixed with one drop of 
the donor's blood. Tn the second, nine drops of the donor's 
blood are mixed with one of the recipient's in the same way. 
The two mixtures are agitated or stirred to insure thorough 
mixing, and allowed to stand for fifteen minutes. At the end 
of that time a drop is placed on a slide and examined under 
the microscope. Agglutination in the first mixture indicates 
that the recipient's plasma is incompatible with the donor's 
corpuscles and would possibly hemolyze them as explained 
before. Agglutination in the second mixture indicates that the 
donor's plasma is incompatible with the recipient's corpuscles. 
The absence of agglutination shows that the two bloods are 
compatible and that hemolysis will not occur. Agglutination 
may occur without hemolysis, but hemolysis does not occur 
without agglutination. 
The amount of blood required for this test can easily be 
obtained by pricking the finger and allowing the blood to drop 
directly into the citrate solution. The drops may not be of the 
same size but that does not matter as the mixture is only 
approximate and is sufficiently accurate for clinical purposes. 
18. Blood Proteins: (a) Fibrinogen. Some citrated or 
oxalated plasma is obtained by sedimenting the corpuscles with 
the centrifuge. The fibrinogen is precipitated by half-satu- 
ration with sodium chloride. Other globulins require full satu- 
ration with sodium chloride for precipitation. 
(b) Serum Globulin. After filtering off the precipitated 
fibrinogen in the last experiment, an equal volume of a saturated 
solution of ammonium sulphate is added to the filtrate. Observe 
that serum globulin is precipitated by half saturation with am- 
monium sulphate. 
(c) Serum Albumin. The filtrate from the last experiment 
is completely saturated with ammonium sulphate by adding the 
crystals until no more can be dissolved. Observe that serum 
albumin is precipitated. 44 
Laboratory Exercises in Physiology. 
19. Coagulation of Blood: Coagulation of the blood may 
be prevented by the following methods: 
1. Receiving the blood into a vessel surrounded by a freezing 
mixture of ice and salt. 
2. Receiving blood into a saturated solution of MgSO4 in the 
proportion of three of the former to one of the latter. 
3. Receiving the blood into a solution of an oxalate to form a 
mixture of 0.2 per cent or more of the oxalate. 
4. A mixture of 0.25 per cent or more of sodium citrate. 
5. A mixture of at least 0.3 per cent of sodium fluoride. 
6. Hirudin in the proportion of 0.001 gram, to 8 c. c. of blood. 
This prevents extravascular clotting and also destroys the 
coagulability of the blood when injected into an animal without 
having any toxic effect. Hirudin is a proteose body prepared 
from the heads of leeches and acts as an antithrombin. 
7. The intravenous injection of commercial peptone, in the 
proportion of 0.3 to 0.5 gram, per kilogram of body weight, 
usually, but not invariably, destroys the coagulability of the 
blood. Sometimes it fails to lessen the coagulability. Peptone 
does not have any effect on extravascular clotting when shed 
blood is received into a peptone solution. 
Magnesium Sulphate Plasma. Dilute a fresh mixture of 3 
parts of blood in 1 part of sat. sol. MgSO4 with 4, 8, 10 and 12 
parts of distilled water. Observe that dilution with four vol- 
umes of water is not sufficient to cause clotting; that clotting 
occurs when fresh "salted plasma" is diluted with 8 or 10 parts 
of water. If the blood has been standing on ice for 24 hours or 
more, a greater dilution may be required. If the corpuscles are 
centrifugalized from such blood the clear plasma may not clot 
on dilution or even on the addition of tissue extracts. 
Apparently the MgSO4 interferes with the interaction of the 
fibrin factors, and if the mixture is allowed to stand for 24 hours 
or more, some essential factor is precipitated. 
O rotated Blood. A 0.5 per cent oxalated blood is obtained by 
receiving 3 parts of blood into 1 part of 2 per cent ammonium 
oxalate, and this mixture is used for the following experiments: 
(a) Observe that dilution, even with 15 or 20 parts of 
distilled water, does not cause clotting. 46 
Laboratory Exercises in Physiology. 
(b) The addition of 6-8 drops of 2 per cent CaCl2 to 5 c. c. 
of oxalated blood is not sufficient to recalcify the blood, but 10 
or more drops cause clotting. 
(c) Tissue extracts, prepared by grinding up liver and 
muscle in physiological salt solution after these tissues have been 
washed free from blood, do not cause clotting. 
(d) Serum causes clotting wffien added in sufficient amounts 
(2-5 c. c. of serum to 5 c. c. oxalated blood). The comparison 
between the serum and tissue extracts can best be shown by par- 
tially, but not completely, recalcifying the blood by adding 3 or 
4 drops of 2 per cent Ca Cl2. It is clear from these experiments 
that soluble oxalates prevent coagulation solely by removing the 
calcium in the formation of an insoluble oxalate. The dilution 
of blood or the addition of tissue extracts can not cause clotting 
unless the blood is recalcified. 
Citrated Blood. While 0.25 per cent of sodium citrate will 
prevent blood from clotting, a 0.5 per cent mixture is used for 
the following experiments: 
(a) Dilution with distilled water does not cause clotting. 
This indicates that the citrate does not prevent cellular dis- 
integration or any interaction between the fibrin factors. 
(b) 'The addition of 2 drops of 0.25 per cent sol. Ca Cl2 to 
5 c. c. of 0.5 per cent citrated blood does not cause clotting, but 
the addition of 4 or more drops causes a clot to form. From 
this it is clear that citrated blood is much more easily recalcified 
than the oxalated blood. In the latter an insoluble precipitate of 
calcium oxalate is formed, while the citrate forms a double salt 
with the calcium of the blood, viz., calcium sodium citrate which 
is not insoluble but non-ionizable. 
(c) Tissue extracts do not cause citrated blood to clot unless 
enough of the extract is added to recalcify the blood. 
(d) Blood serum causes coagulation by the excess of thrombin 
contained in it. 
Fluoride Blood. A 0.6 per cent sodium fluoride mixture is 
obtained for the following experiments by receiving 3 parts of 
blood directly from the blood vessel into 1 part of a 2.4 per cent 
sodium fluoride solution. 48 
Laboratory Exercises in Physiology. 
(a) Dilution with water does not cause clotting. 
(b) The addition of an excess of CaCl2 (10 drops of 2 per 
cent CaCl2 to 5 c. c. fluoride blood) may cause clotting. The 
addition of 5-6 drops of 2 per cent CaCl2 may be sufficient to re- 
calcify 5 c. c. of blood by combining with all the excess of 
fluoride to form an insoluble calcium fluoride, but clotting does 
not occur. 
(c) The addition of tissue extracts does not cause clotting. 
(d) The addition of blood serum in sufficient amount (2-5 
c. c. serum to 5 c. c. blood) causes clotting. 
(e) Recalcified fluoride plasma is prepared by adding 6 
drops of 2 per cent CaCl2 to 10 c. c. of fluoride blood. The above 
tests are then repeated. Observe (1) that dilution with water 
now causes clotting; (2) that tissue extracts now cause clotting 
as well as blood serum. 
These experiments indicate that sodium fluoride prevents 
coagulation partly by decalcifying the blood, and in addition, 
by preventing cellular disintegration and the liberation of a 
thromboplastic substance (thrombokinase). 
Peptone Plasma. The injection of commercial peptone does 
not always destroy the coagulability of blood, but if peptone 
plasma can be obtained the following experiments may be per- 
formed : 
(a) Dilute with 4, 8, and 12 volumes of distiled water and 
record the result. Dilution usually causes peptone plasma to 
clot, showing that the interaction between the precursors of 
fibrin is prevented in some way. 
(b) The addition of CaCl2 alone does not cause it to clot. 
(c) The addition of tissue extracts, in the same propor- 
tions that were used in the previous experiments causes a clot. 
It is clear from this experiment that the formation of thrombin 
has been prevented in some way. The thromboplastic sub- 
stance (thrombokinase) appears to be inactive or unavailable 
for some reason. 
(d) To 5 c.c. of peptone plasma add blood serum in increas- 
ing quantities, starting with 1 c.c. and increasing 1 c.c. at a time 
until coagulation occurs. Record the result. Observe that 
much more of the serum is required than of the tissue extracts. 50 
Laboratory Exercises in Physiology. 
The reverse is true of fluoride and citrated blood. The fact 
that an excessive amount of serum, which contains thrombin, 
has to be added indicates that peptone plasma probably con- 
tains an antithrombin. 52 
Laboratory Exercises in Physiology. 
DIGESTION. 
20. Comparative Anatomy of the Alimentary Canal: Copy 
the table showing the proportion of the length of the body to the 
length of the alimentary canal in the cat, dog, horse, pig, ox, 
sheep and in man. 
Copy the tabular statement showing the relative capacities 
of the stomach, small intestine and large intestine in the dog, 
horse, sheep and pig. 
Copy the tabular statement showing the length of the small 
intestine and the length of the large intestine in the cat, dog, 
rabbit, and pig; also in man at birth and in an adult man. 
Examine the anatomical specimens of the alimentary tract. 
Examine particularly the stomach, the pyloric sphincter, the 
wall of the antrum, position of the transverse band, fundus, etc. 
From these observations briefly state your conclusions con- 
cerning (1) the relation of the nature and amount of food 
consumed to the length and capacity of the alimentary canal; 
(2) to the length of the small and large intestine, respectively; 
(3) state in a broad way the function of the stomach, small 
and large intestine, respectively. 
Examine the skulls of rodents, carnivora, herbivora and 
omnivora showing the articulation of the mandible with the 
skull; also the kinds and arrangement of the teeth. Examine 
the temporary and permanent teeth in man. 
21. Influence of the Mechanical Comminution of Food on 
the Chemical Changes of Digestion: 1. Artificial Digestion. 
Some raw egg white is stained deeply with ammonia carmine 
and then slowly coagulated by heat so that the stain is fixed 
in the coagulum and can only go in solution in an artificial 
peptic digest as proteolytic digestion occurs. 
The coagulated egg white is divided into two equal por- 
tions by weight. One part (A) is cut up into large cubes; 
the other part (B) is finely ground up by passing it through 
a meat-chopper. Each part is then put in a beaker containing 
the same amount of pepsin and 0.4 per cent HC1. Both beakers 
are kept in the incubator at 38° C. for 4 or 5 hours. Contrast 
the amount of digestion in the twyo beakers by comparing the 54 
Laboratory Exercises in Physiology. 
depth of coloring with the carmine that has passed in solution 
and record the result. 
2. Natural Digestion. Four dogs are fasted long enough 
to make certain that their stomachs are empty. The first two, 
A and B, are each given 100 grams of lean meat cut in large 
cubes so that the food has to be masticated preparatory to 
deglutition. 
Two other dogs, C and D, are each given 100 grams of lean 
meat that has been finely ground up by passing it through 
the meat-chopper. 
At the end of three hours, a hypodermic injection of 0.2 
of a grain of apomorphine is given to dogs A and C, and the 
vomited meat, which is promptly and completely expelled, is 
collected in a pan. 
At the end of four, or four and one-half hours, the meat 
is recovered from the stomachs of the dogs B and D in the 
same way. 
Contrast the amount of food that remains undigested in 
each case after three and after four hours. Write a brief de- 
scription of the appearance of the meat and state how thorough 
comminution favors digestion. 
22. The Chemical Changes of Gastric Digestion: The meat 
recovered after 3 and 4 hours of natural gastric digestion in 
the preceding experiment is suspended in water and examined for 
the different stages of proteolysis as follows: 
(a) Acid albumin (neutralization). 
(b) Primary proteoses (half saturation with (NII4)2SO4). 
(c) Secondary proteoses (complete saturation with (NH4)2 
SO.). 
(d) Peptones (Biuret test after removing proteoses). 
Record the result. Observe that all occur. 
Tn the same way some of the natural digest of 3 and 4 
hours, respectively is titrated against NaOII and the 
total acidity, free and combined TTC1 are determined. Record 
the result. 
23. Conditions Affecting Peptic Digestion: The same 
amount of fibrin stained with ammonia carmine is placed in 
each of the following test-tubes and the artificial digests are 
kept in the incubator for several hours: 56 
Laboratory Exercises in Physiology. 
(a) Pepsin in 0.4 per cent HC1 (control). 
(b) Pepsin in neutral solution. 
(c) 0.4 HC1 alone without pepsin. 
(d) Pepsin + 0.4 p. c. HC1 boiled. 
(e) Pepsin + lactic acid (equivalent to 0.4 per cent 11C1). 
(f) Pepsin + 0.1 per cent HC1 (hypoacidity). 
Observe the depth of color from the dissolved carmine and 
record the results. 
Mett's tubes may be used to show that the amount of enzyme 
influences the rapidity of digestion. * 
24. Conditions Affecting the Action of Rennin: Test tubes 
are prepared having the same amounts of rennin and milk, 
as follows: 
(a) Rennin + milk (control). 
(b) Boiled rennin + milk; enzyme destroyed. 
(c) Neutralized rennin + milk. 
(d) Rennin milk diluted with equal volume of water. 
(e) Reniiin + human milk (very fine curd). 
(f) Rennin fl- citrated milk. Clots on addition of CaCl2. 
The digest may be boiled before adding CaCl2. 
25. Conditions Affecting the Digestion of Curdled Milk: 
Small beakers are prepared each having the same amount of 
milk (100 c. c.) plus pepsin in 0.4 p. c. HC1. 
(a) Milk plus pepsin in IIC1 (control). 
(b) Milk diluted with an equal part of water. 
(c) Milk diluted with an equal part of starch water. 
(d) Milk mixed with bread crumbs. 
(e) Milk mixed with cracker crumbs. 
Observe and record the result in each case. What is the 
advantage of starch water over plain water as a diluent for 
milk in promoting gastric digestion? How do bread or crack- 
er crumbs favor the digestion of milk? 
26. Conditions Affecting the Acidity of the Gastric Juice: 
Compare the amount of total, free and combined HC1 in the 
stomach content of several different dogs as determined by 
titration after a test-meal of cracker crumbs suspended in 
water and removed by a stomach tube at the end of one hour. 
Contrast the above control in each case with the determina- 58 
Laboratory Exercises in Physiology. 
tion of total, free and combined HC1 after feeding (a) with 
milk; (b) with egg white; (c) with meat after 2 and 4 hours, 
respectively. How do foods rich in proteins compare with a 
test-meal of crackers or bread crumbs in exciting the flow of 
HC1? 
How does meat compare with egg-white in stimulating the 
secretion of HC1 when relatively the same amount of protein 
is given, diluted to the same volume with water? 
How does milk compare with the test meal of cracker crumbs 
in water? 
27. The Influence of Meat Extractives on the Secretion of 
Gastric Juice: Two animals (dogs or cats) may be anesthe- 
tized and have the cardiac and pyloric orifices of the stomach 
tied off after a certain amount (from 75 to 200 c.c.) of water 
has been introduced into the empty stomach of one. and the 
same amount of bouillon into the empty stomach of the other. 
The animals may be kept lightly under ether and at the end 
of one hour the fluid is collected from each animal, measured 
and titrated against ™ NaOH. Record the result. Observe 
that very little (not more than 5 per cent) of the water is 
absorbed and it practically does not cause any secretion of 
HC1; that the amount of fluid is increased in the stomach con- 
taining bouillon and the acidity is three or four times greater 
than that containing water. 
The influence of creatin as a gastric secretagogue may also 
be shown by giving a test-meal of cracker crumbs suspended 
in bouillon and comparing it with the acidity found in a pre- 
vious test-meal of cracker crumbs and water in the same dog. 
The stomach contents removed by a stomach tube in this 
way can only be used for comparisons in acidity. The amount 
of secretion can not be determined as some fluid escapes through 
the pylorus. 
28. Psychic Secretion of Gastric Juice: Two cats or dogs 
are starved for one or two days until they show evidence of 
hunger. One of them is then placed in a cage with a false 
floor made of wire mesh. Meat or fish is placed beneath the 
wire mesh so that the animal can see and smell the food 
but can not eat it. After several hours of excitement caused 60 
Laboratory Exercises in Physiology. 
in this way, both animals are placed under an anesthetic and 
a cannula is introduced into the stomach of each through the 
pyloric splineter from the duodenum. The fluid contained in 
each stomach is collected, measured and titrated. Record the 
result. 
If the sight and odor of food caused much excitement, there 
is a decided increase in the quantity and in the acidity of the 
fluid in the stomach of that animal as compared with the con- 
tent of the other animal starved for the same time but not 
excited by food. 
29. Mechanical Stimulation of the Gastric Mucosa: When 
inert and insoluble substances, such as short pieces of a wax 
taper, are introduced into the stomach of a dog with a definite 
amount of water, observe that the fluid drawn off from the 
stomach at the end of a half-hour or one hour shows very little 
increase in total acidity and no free HC1. Record the result and 
contrast with the secretion caused by cerebral excitement; also 
with that caused by the secretagogue creatin in the preceding 
experiment. Compare the result of mechanical stimulation in 
this experiment with the result obtained in the next one. 
30. The Influence of Secretory Nerve Fibers on the Gastric 
Glands: One vagus nerve, preferably the right, may be cut, 
with the precautions of asepsis, three days before this experi- 
ment is performed. This allows the cardio-inhibitory fibers to 
degenerate but does not interfere with respiration. The secre- 
tory nerve fibers to the gastric tubules do not degenerate as 
rapidly as the cardio-inhibitory fibers and therefore they may 
be stimulated by stimulating the peripheral end of the nerve 
with a tetanizing current three or four days after cutting it. 
In this way any disturbance of the circulation is avoided. 
Or, both vagi may be stimulated by single induction shocks 
at intervals of one second without preliminary section of the 
nerve. These are sufficient to excite the gastric glands to 
activity without lowering the blood-pressure. 
A gastric fistula is produced and the two openings tied off in 
a fasting dog; the wound is closed, and the stomach is washed out 
with warm water. Mechanical stimulation may be applied for 
15 minutes by introducing a feather or several wax tapers. Any 62 
Laboratory Exercises in Physiology. 
secretion caused by the mechanical stimulus is usually alkaline 
from mucus. 
After again washing out the stomach, the animal is placed 
in a special holder so that the gastric juice drains from the 
cannula as it is formed. The secretory fibers of the vagus are 
stimulated by one of the methods mentioned above for twenty min- 
utes. Observe the long latent period. Secretion does not begin 
until the nerves have been stimulated for 6 or 7 minuts. After 
secretion begins, the gastric juice is collected for 5 minute 
periods. Make a protocol of the experiment showing the latent 
period and the amount secreted for the different periods. 
If secretion does not occur, on account of the anesthesia and 
surgical shock, a small does of pilocarpine may be given intra- 
venously and the stimulation applied as before. If this succeeds 
an injection of atropine may subsequently be made and the stimu- 
lation applied as before. Pilocarpine stimulates the secretory 
fibers while atropine paralyzes them. 
The gastric juice collected in this experiment is titrated for 
total acidity and free HC1. The digestive enzymes may be shown 
by adding some of it to milk and another part to fibrin, keeping 
the test-tubes at the bodily temperature. 
Copy a protocol of the secretion obtained in various ways 
from a dog with a fistula of the stomach by Pawlow's method. 
31. The Digestion of Living Tissues; Auto-digestion: Five 
frogs have their brains pithed and are given enough curare to 
produce motor paralysis. They are then suspended so that one 
leg of one is kept in a beaker containing 0.2 per cent IIC1; an- 
other in a beaker containing pepsin and 0.2 per cent IIC1; the 
third in a neutral solution of trypsin; the fourth in a solution 
of trypsin in 1 per cent Na., CO3 and the fifth in 1 per cent 
Na, CO3. 
All of the beakers are kept in a water bath at 37°C for 3 or 4 
hours. Observe that pepsin and IIC1 cause complete digestion 
of all the soft tissues with which the digest comes in contact; 
that the acid or the alkali alone, or the trypsin cause maceration 
of the skin, but not complete solution of the tissues. Record 
the result. 
The bladders of dogs filled with pepsin and HC1 show the 64 
Laboratory Exercises in Physiology. 
same digestion, even to the extent of perforation. The animals 
were narcotized bnt the circulation was normal. Can the circu- 
lation of the alkaline blood and lymph explain why the living 
stomach does not digest itself, as the wall of the dead stomach 
is digested when removed during active digestion and kept at 
the bodily temperature for a few hours ? 
Antiferments may be shown by adding a small amount of a 
glycerine extract of tape-worms to an artificial digest of fibrin 
in pepsin-HCl and also to an artificial digest of fibrin in trypsin 
and Na2 CO3. These are compared with control digests and 
the influence of the antiferment in restraining proteolytic diges- 
tion can be seen. This explains how intestinal parasites and 
the intestinal mucosa escape digestion. The bearing of auto- 
digestion upon the pathogenesis of gastric and duodenal ulcers 
is obvious. 
32. The Action of Secretin on the Flow of Pancreatic Juice: 
A solution of secretin is extracted from the intestinal mucosa 
as follows: The mucous membrane from the dudoenum and 
upper 3 or 4 feet of the jejunum is scraped off and chopped up 
finely. It is then mixed with washed sand and ground up in a 
mortar with 150 c.c. of 0.2 per cent HC1. It is allowed to stand 
until a short time before the experiment, when it is fiiltered 
through gauze. It is then carefully, neutralized by adding 
NaOH. After bringing it to the neutral point a few drops of 
dilute acetic acid are added to give it a faintly acid reaction. 
An extract from the lower end of the ileum is prepared in 
the same way. 
A dog is anesthetized, the abdomen opened and cannulas are 
introduced into the common bile duct and into one of the pan- 
creatic ducts. The cystic duct is ligated and the number of 
drops falling from each of the cannulas is counted for two 
periods of two minutes each. 
An intravenous injection of 10 c.c. of the extract from the 
ileum is then made and the number of drops counted again. 
Record the result. 
The same amount of the extract of the duodenum and jeju- 
num is then injected intravenously. The number of drops is 
again counted and recorded. 66 
Laboratory Exercises in Physiology. 
Is secretin contained in the mucous membrane of the lower 
end of the ileum as it is in the upper part? 
Is the flow of bile stimulated to the same extent as the flow 
of pancreatic juice? 
How long does the flow continue? An injection of dilute 
HC1 (0.2 per cent) may be made into the lumen of the duodenum 
when the flow of pancreatic juice returns to the normal after 
the intravenous injection of secretion. The number of drops 
is counted again and recorded. 
33. Pancreatic Digestion: Some fresh pancreatic juice is 
collected from a cannula tied in the pancreatic duct of another 
dog. A fistula of this kind may be used to collect the pancreatic 
juice before it enters the intestine. Describe its appearance and 
give its reaction. 
A. Action on Starches. 
1. Add 5 or 10 drops to some 0.7 per cent solution of boiled 
starch at the temperature of the body and test for sugar every 
15 or 20 seconds until it appears. Record the result. Boil with 
Barford's reagent and observe that dextrose is not formed. 
2. Repeat this experiment using a suspension of raw starch 
that has been ground up in a mortar, or use raw potato and 
compare with cooked potato. Record the result. 
3. Repeat the experiments using diazyme, a commercial ex- 
tract of pancreas, instead of the pancreatic juice. 
4. Repeat 1 and 2 using saliva in the same amount, instead 
of pancreatic juice, and compare the time required for the 
appearance of maltose. 
5. Add 10 drops of pancreatic juice to each of two test-tubes, 
A and B. To the tube B add 10 drops of a 3 per cent solution 
of bile salts. Now add a lukewarm solution of boiled starch to 
each and, after waiting 15 to 30 seconds, add 1 or 2 drops of a 
weak iodine sol. (Lugol's sol.). Invert the tubes repeatedly and 
observe the rapid change from starch to erythrodextrin and 
achroodextrin in B as compared with A. This is shown by the 
change in color from blue to violet, then to red and finally the 
disappearance of all color with the formation of the final pro- 
ducts of starch digestion. 68 
Laboratory Exercises in Physiology. 
What is the influence of bile on the pancreatic digestion of 
starches? 
Are the bile salts necessary to activate the amylopsin or is the 
pancreatic amylase capable of digesting starches before it comes 
in contact with bile? 
How do co-ferments differ from activators? 
B. Action on Fats. Some milk is boiled to kill the acid- 
producing bacteria in it. A few drops of phenolphthalein are 
added and enough NaOH is then cautiously added to give it 
a distinct and permanet pink color. The degree of fat digestion 
will be indicated by the amount of fatty acids liberated as de- 
termined by titration with ?CNaOH. 
1. Take the same amouPt of lukewarm, alkaline milk con- 
taining phenolphthalein as prepared above in each of two test- 
tubes, A and B. To A add 10 drops of fresh pancreatic juice; 
to B add 10 drops of pancreatic juice that has been boiled in a 
little water in another tube. Place both tubes in a water-bath 
at 37°C for 3 or 4 minutes. Observe the rapid disappearance 
of the pink color in A. Then add ™ NaOH from a burette or 
graduated pipette to each until a permanent pink color is again 
established. Record and explain the results. 
2. Take three test-tubes labeled A, B and C. To A and B 
add 10 drops of fresh pancreatic juice; to B and C add 10 drops 
of a 3 per cent sol. of bile-salts. Add the same amount of 
alkaline milk containing phenolphthalein, as prepared above, to 
each one and place all of the test-tubes in a water bath at 37°C 
for 4 or 5 minutes. At the end of that time boil each tube to 
stop the enzyme action. Now titrate N NaOH into each one 
until a permanent pink color is produced and record the result. 
Can the pancreatic lipase, steapsin, act without bile-salts? 
What is the influence of bile-salts upon the pancreatic diges- 
tion of fats? 
Can bile-salts alone cause the hydrolytic cleavage of neutral 
fats? 
Do the bile-salts act as activators or as co-ferments? 
3. The influence of emulsification in promoting the digestion 
of fats by the pancreatic lipase may be shown by the following 
experiment: The same amount (3 c.c.) of neutral cottonseed 70 
Laboratory Exercises in Physiology. 
oil is taken in each of two test-tnbes, A and B. Some distilled 
water (about 5 or 6 c.c.) is added to A and the same amount of 
a solution of ivory soap is added to B. Both tubes are shaken 
well. An emulsion is formed in B while the fat separates at the 
top of A. 1 c.c. of fresh pancreatic juice is then added to each 
and the tubes are placed in a beaker of lukewarm water for 10 
minutes. At the end of that time the tubes are boiled to stop 
further digestion. A few drops of phenolphthalein are added 
and ™ NaOH sol. is titrated into each. Record the result. 
How does emulsification aid fat digestion by steapsin? 
4. Repeat the experiments 1, 2 and 3, using a fresh solution 
of holadin instead of the pancreatic juice. Holadin is a com- 
mercial extract of pancreas containing the pancreatic lipase. 
Record the result and compare with the results obtained in 1 
and 2. 
C. Action on Proteins. To each of three test-tubes, A, B 
and C, add 15 drops of fresh pancreatic juice obtained from a 
cannula tied in the pancreatic duct. To B add some scrapings 
of the intestinal mucosa, and to C add the same after it has 
been boiled. Fill each tube with water and add the same amount 
of fibrin stained with carmine to each tube. Keep all of the 
tubes in the incubator at 38°C for several hours and then com- 
pare the degree of digestion by the depth of the color from 
carmine in solution in each tube; also by the amount of fibrin 
that remains undigested. Explain the difference. 
Is the proteolytic enzyme secreted by the pancreas in active 
form, or is it activated by something contained in the intestinal 
mucosa ? 
What is the activator of trypsinogen and what is the nature 
of its action ? 
34. Conditions Influencing Tryptic Digestion: Seven test- 
tubes are labeled A, B, C, D, E, F and G. The same amount 
(1 c.c.) of an extract of pancreas or a solution of pancreatin is 
added to each one except G. A is boiled; B and C are neutral- 
ized using litmus as an indicator; 1 c.c. of a solution of bile-salts 
is added to C; D is made faintly acid with dilute lactic acid; E 
is made acid with 0.2 per cent HC1. The tubes A, B, C, D and E 72 
Laboratory Exercises in Physiology. 
are then filled with distilled water. The tubes F and G are 
filled with 1 per cent Na2 C03. The same amount of fibrin 
stained with carmine is then added to each tube and the tubes 
are kept in the incubator for 3 or 4 hours. At the end of that 
time the tubes are inverted and the depth of color from carmine 
in solution is compared to determine the degree of digestion in 
them. Record the results in answering the following questions: 
1. Does boiling destroy trypsin (tube A) ? 
2. Can trypsin act in a neutral medium (B) ? 
3. Is the action of trypsin intensified by bile-salts as much 
as the other pancreatic enzymes (C) ? 
4. Can trypsin act in the presence of an organic acid (D) ? 
5. Can trypsin act in the presence of dilute HC1 (tube E) ? 
Trypsin is destroyed by free but not by combined HC1. 
6. Is the digestion in an alkaline medium equal to that of 
the pancreatic juice (tube F) more active than that in a neutral 
medium (tube B) ? Compare tubes F and G and allow for the 
solution of the carmine by the Na2CO3 alone as seen in tube G. 
35. The Peptonization of Milk: The same amount of milk 
(100 c.c.) is taken in each of six beakers A, B, C, D, E and F. 
To A and B add pepsin dissolved in 0.2 per cent IIC1; to C and 
D add pancreatin or extract of pancreas and enough sodium 
bicarbonate to give an alkaline reaction; to E and F add pepto- 
genic milk powder. 
Place all six beakers in the incubator at 37°C. At the end of 
15 minutes take A, C, and E out and boil to stop further diges- 
tion. At the end of 30 minutes remove B, D and F and boil. 
Observe the undigested curd in A and B. Why is it im- 
practicable to peptonize milk with pepsin and HC1? 
Apply the following test to C and E to determine the amount 
of caseinogen which remains undigested, comparing the result 
in each case with a control test in which the same amount of un- 
digested milk is treated in exactly the same way. 
1. Dilute some of the milk in a test tube with four parts of 
warm water and add dilute acetic acid from a pipette until the 
caseinogen is precipitated in curds. The mixture is kept warm 
by passing it through the flame from time to time while adding 
the acid. 74 
Laboratory Exercises in Physiology. 
Repeat this test with some of the milk from the beakers D and 
F, using the same degree of dilution and cautiously add dilute 
acetic acid until a fine precipitate forms. An excess of acid 
should be avoided as it re-dissolves the precipitate. 
Contrast the amount and particularly the character of the 
precipitate in C and E with that in the control. Observe the 
further change in D and F. 
What change takes place in milk after peptonization for 10 
or 15 minutes with the active proteolytic enzyme extracted from 
the pancreas? 
How does this change influence the digestibility of milk when 
it is subsequently taken into the stomach? 
What is the action of rennin on peptonized milk? 
2. In which ones of the six beakers has a bitter taste devel- 
oped and what is the cause of it? How can this unpleasant 
taste be prevented when milk is properly peptonized? 
3. Apply the biuret test and observe the pink or rose color 
of proteose in the peptonized milk as compared with the bluish 
violet color of undigested milk. The same amount of milk is 
taken from the beakers C, D, E and F in each of four test tubes 
and an equal amount of undigested milk is taken to a fifth tube. 
An excess of 15 per cent NaOH is then added. A few drops of 
1 per cent CuSO4 are then added until the same depth of color 
is obtained in each tube. 
36. Bile. Hay's Test for Bile Salts. Take the same amount 
of water in each of three test tubes, A, B and C. Add 10 
or 15 drops of bile to A and the same amount of a sol. of 
bile salts to B. Keep C as a control. Sprinkle a few particles 
of powdered sulphur on the surface of each. Observe that the 
sulphur quickly sinks to the bottom of A and B but floats on the 
surface of the water in C. Bile salts lower the surface tension 
of fluids in which they are dissolved and for this reason the 
sulphur sinks to the bottom. It can not be wetted in the tube 
C. This is a very delicate test and it can detect 1 part of bile 
salts in 100,000 of water. It is a very satisfactory test for 
detecting bile salts in the urine when the outflow of bile into 
the intestine is obstructed with consequent absorption into the 
blood. 76 
Laboratory Exercises in Physiology. 
37. The Gmelin-Heintz Test for Bile Pigments: A few 
drops of bile, or a weak solution of bile, are placed on a white 
plate and allowed to come in contact with impure HNO3 con- 
taining HNO2. Observe the play of colors at the line of con- 
tact from the oxidation of the bile pigments: green, blue, violet, 
red and yellow. 
38. Interaction between the Bile and the Chyme: Take 
four test-tubes A, B, C and D. Add 5 c.c. of 0.2 per cent HC1 
to A and B and the same amount of a filtered peptic digest to 
C and D. To A and C add a solution of bile salts until a pre- 
cipitate forms; to B and D add fresh bile. Observe a slight 
precipitate of bile salts in A and a heavier precipitate of mucin 
and bile salts in B. In C the products of digestion are pre- 
cipitated; in D the products of digestion are precipitated, in 
addition to the action of the acid on the bile, giving the heaviest 
precipitate of all. When an excess of bile or of the solution 
containing bile salts is added, most of the precipitated products 
of digestion are re-dissolved. 
39. Solvent Action of Bile on Insoluble Soaps: Take 5 c. c. 
of a soap solution in each of two test-tubes, A and B. Add a 
drop or two of a CaCl2 sol. to each and observe the formation 
of an insoluble calcium soap. To A add a solution of bile salts 
and to B add the same amount of distilled water. Shake the 
tubes and observe that the insoluble calcium soap is dissolved by 
the bile salts in the tube A. 
40. Solvent Action of Bile on Fats: Moisten two filter 
papers with a 5 per cent solution of bile salts on funnels A and 
B. Moisten two other filter papers with water on funnels C 
and D. Pour neutral cottonseed oil on the filter papers in the 
funnels A and C and rancid fish oil in B and D. Observe that 
the solvent action of bile on neutral fats allows some of the oil 
to pass through the filter paper in A, while the greater solvent 
acid for fatty acids makes it possible for a larger amount of the 
rancid fish oil to pass through the filter B in the same time. 
None of the oil can pass through the filters C and D while 
they are wet with water. 
41. Intestinal Digestion of Proteoses by Erepsin: A 0.5 78 
Laboratory Exercises in Physiology. 
per cent of commercial "peptone" is prepared and the same 
amount is taken in each of three beakers, A, B and C. To A 
and B the same amount of the scrapings from the intestinal 
mucosa is added. B is boiled to destroy the enzyme. 
A and B are placed in the incubator at 37°C for several 
hours. At the end of that time the following tests are applied 
to some of the digest from A and B', respectively, comparing 
each one with a control test from the original peptone solution 
in C: 
1. Apply the biuret test. Observe that proteoses disappear 
from A, containing active erepsin, but remain in B. 
2. Hoffman's test for tyrosin. Add Millon's reagent and 
boil. Observe the deep red color in the solution in A as com- 
pared with the light dull red color of the precipitate in B and C. 
The latter is due to the proteins while the former is due to 
tyrosin. 
3. Apply the tryptophan test by adding bromine water to 
some of each of the solutions. This is only obtained with the 
digest A. 
If these reactions do not show after the digest has been in the 
incubator four hours, the digestion should be continued for 24 
hours and the tests repeated. 
42. Inverting Enzymes of the Intestinal Mucous Membrane: 
Scrapings or shreds of the intestinal mucosa are put in 1 per 
cent solutions of the following: (a) Sucrose; (b) maltose; (c) 
lactose and (d) dextrin. A portion of each digest is then 
boiled to destroy the enzymes, and all of the digests, boiled and 
unboiled, are placed in the incubator for several hours. 
A portion of each digest is then boiled with Barfold's solution 
and a positive reaction shows the formation of dextrose by the 
inversion of the di-saccharides. 
43. Movements of the Stomach and Intestines: A cat is 
fed with finely chopped meat mixed with boiled corn meal, four 
hours before the experiment. The cat is anesthetized through 
a tube tied in the trachea and is fastened in a special holder so 
that it can be completely immersed in Ringer's solution. The 
Ringer's solution is kept at the temperature of the body and 80 
Laboratory Exercises in Physiology. 
prevents contact of the viscera with the air when the abdomen 
is opened. 
A long median incision is made in the abdominal wall and the 
stomach, small intestine and large intestine exposed to view while 
these viscera are kept submerged beneath the Ringer's solution. 
If the normal movements do not occur they may be set up by 
stimulating the vagi in the neck with induction shocks at in- 
tervals of one second, the stimuli being interrupted from time 
to time. 
Observe where the movements begin in the wall of the stomach 
and the way in which they pass to the pylorus. Observe par- 
ticularly the change at the transverse band and in the wall of 
the antrum; also the succession of contraction waves. 
Observe the rhythmic and the peristaltic waves in the wall of 
the small intestine and the distance the latter travel. 
Observe the reversed peristalsis or antiperistaltic waves in the 
ascending colon. 
If these movements only occur from electrical stimulation of 
the accelerator fibers in the vagi, observe how they subside when 
this artificial stimulation is discontinued from time to time. 
White an account of the observations made in this experiment. 82 
Laboratory Exercises in Physiology. 
RESPIRATION. 
CHANGES IN THE AIR FROM RESPIRATION. 
44. Composition of the Atmosphere: (a) Percentage of 
Oxygen: A definite amount (100 c.c.) of the air of the labo- 
ratory is measured in a Hempel burette. It is then forced into a 
Hempel gas pipette over a concentrated solution of pyrogallic 
acid and caustic potash. This absorbs the oxygen and the CO,, 
but the amount of the latter is so slight in this case that it may 
be disregarded. 
The air is then forced back into the burette, the pressure 
equalized, and the volume noted. The diminution in volume in 
100 c. c. of air indicates the volume per cent of oxygen in the 
air of the room. Record the result. 
(b) Percentage of CO2 in the Atmosphere: Low percentages 
of CO, can not be determined by the slight reduction in a small 
volume of air after absorption by a caustic alkali in a gas pipette. 
The most accurate method is to absorb the CO, from a large vol- 
ume of air by shaking with lime water, or baryta water, of 
definite strength, and determine the consequent reduction in 
alkalinity by titrating against a weak standard acid of definite 
strength (Pettenkofer's method). 
A convenient method is that suggested by Dr. Angus Smith, 
which has been standardized by comparison with Pettenkofer's 
method. It depends upon the amount of air required to produce 
turbidity in lime water. The larger the amount of air required, 
the lower the percentage of CO,; the smaller the volume of air 
required to produce turbidity, the higher the percentage of CO,. 
Bottles of different sizes are unstoppered in a room so that the 
air of the room may mix by diffusion with the air in the bottles; 
into each bottle a small amount (15 c. c.) of freshly filtered 
lime water is placed; the bottles are then stoppered and shaken 
for a few minutes. Distinct turbidity of the lime water in any 
but the largest bottles indicates an excess of CO, above the nor- 
mal (0.03 to 0.04 per cent). Turbidity in the different bottles 
indicates the following percentages: 84 
Laboratory Exercises in Physiology. 
550 c. c. bottle=.03 per cent CO2. 
450 c. c. bottle=.04 per cent CO2. 
350 c. c. bottle=.05 per cent CO2. 
300 c. c. bottle=.06 per cent CO2. 
260 c. c. bottle=.07 per cent CO2. 
225 c. c. bottle=.08 per cent CO2. 
200 c. c. bottle=.09 per cent CO2. 
185 c. c. bottle=.10 per cent CO,. 
100 c. c. bottle=.20 per cent CO2. 
45. Composition of Expired Air: The excess of CO2 and 
the deficiency of O2 may be demonstrated by two very simple 
experiments: 
1. Excess of CO2: Two flasks containing freshly filtered 
lime water are arranged like a Muller valve so that the air in- 
haled into the lungs passes through one flask, while that exhaled 
from the lungs passes out through the lime water of the other 
flask. After a few respirations the lime water in the flask 
through which the exhaled air passed, showed pronounced tur- 
bidity, while that in the other one remained perfectly clear, al- 
though the same volume of air was drawn through it. 
2. Deficiency of Oxygen: Some of the air exhaled from the 
lungs is collected in an inverted flask that was previously filled 
with water. Observe that when a lighted taper is thrust into 
the air of this flask the flame is extinguished at once because 
there is not enough O2 to support combustion. 
46. Analysis of Expired Air: The air exhaled from the 
lungs in ordinary respiration is collected over acidulated water. 
A definite quantity (100 c. c.) of this expired aid is measured 
in a Hempel burette. It is then forced into a Hempel gas pipette 
filled with a very concentrated solution of KOH. It should re- 
main there for some minutes and the pipette should be agitated 
so that the CO2 may be absorbed. The air is then returned to 
the burette; the pressure is equalized and the diminution in vol- 
ume indicates the amount of CO2 present. Record the result 
expressed in per cent by volume. 
The air is then forced into a gas pipette containing a concen- 
trated solution of pyrogallic acid and KOH to absorb the O2 as 86 
Laboratory Exercises in Physiology. 
before. The reduction in volume shows the amount of O2 present 
in expired air. Record the volume per cent as before. 
The respiratory quotient .should be determined from the 
amount of O2 absorbed and the CO2 given off in a considerable 
period of time. In the analysis you have seen, compare the com- 
position of the inspired and expired air and determine the loss 
of O2 and the gain of CO2. What is the ratio between the two, 
i. e., what is the respiratory quotient or co-_ ? 
Os 
47. Analysis of Supplemental Air: The exhaled air used 
for analysis in the preceding experiment represents the tidal air 
or that moved in and out of the lungs with each ordinary respira- 
tion. 
The stationary or alveolar air contains a higher percentage of 
CO2 than the tidal air. A part of the air which is stationary in 
the lungs with ordinary respiration may be collected by making 
several forced expirations after ordinary expirations; the air is 
forced through a long piece of rubber tubing (12 feet in length) 
with a T tube near the mouth; from- the side arm of the T tube 
some of the supplemental air is collected over acidulated water 
and analyzed by the same method as in the last experiment. 
Record the result. 
48. Presence of Organic Matter in Expired Air: Two 
Woulff bottles, containing some colorless H2SO4, are arranged 
so that the inspired air is drawn in through one and the expired 
air is forced out through the other. After one breathes through 
these bottles for some time (15 to 20 minutes or more), observe 
that the acid through which the expired air has been passed is 
darkened slightly as shown by the brownish color compared 
with that in the other bottle. This charring is due to the pres- 
ence of organic matter in the expired air. 
The same thing may be shown by the bleaching of a weak alka- 
line solution of potassium permanganate. 
49. Effect of Breathing Air Vitiated by Respiration: A 
number of bell jars containing ten or twelve pigeons (or more if 
necessary) are arranged in series so that the air is drawn from 
one to the other by a Chapman or Crowell suction pump for an 
hour or two. Only the first pigeon gets fresh air; each of the 88 
Laboratory Exercises in Physiology. 
succeeding pigeons breathe the air that has been vitiated by the 
other pigeons ahead of him. When the air moves slowly it is 
found that the air entering the last bell jar (jar A) contains 
about 17 to 18 per cent of O2 and 3.5 to 4 per cent, of CO,. 
Observe the objective symptoms of the pigeon in the last jar 
(A), i. e., the effects of the respiratory impurities given off by 
the other pigeons in the series. Note that the animal not only 
suffers from hypernea and dyspnea, but that there is dullness, 
stupor, closing of the eyes and ruffling of the feathers. 
The air from the last jar (jar A) is divided by means of two 
Y-tubes. Part of it is drawn through a Woulff bottle or cylindri- 
cal percolater placed on its side and containing soda lime to 
absorb the CO2, and then enters jar B containing another pigeon. 
Another part of the air from jar A is drawn through a Woulff 
bottle or percolater containing pumice stone soaked in H,SO4, to 
absorb the organic matter and moisture, and then it passes 
through jar C. 
A third part of the air from jar A is drawn through a con- 
denser consisting of a coil of metal tubing surrounded by a mix- 
ture of ice and salt to reduce the temperature of the air and 
condense some of the moisture before it enters jar D. The tem- 
perature of each jar is taken by thermometers inserted in T- 
tubes at the top of the jars. 
Observe the behavior and objective symptoms of the pigeons 
in jars B, C and D, and compare them with the one in jar A. 
Record your observations. 
1. Does removal of the CO2 (pigeon B) prevent the dullness 
and evident discomfort from which the pigeon A suffers? 
2. Does the high percentage of CO, alone, without the mois- 
ture and organic matter (pigeon C), produce all the symptoms 
seen in pigeon A, or only the dyspnea? 
3. Does the reduction in temperature with its accompanying 
condensation of moisture in jar D, without changing the chem- 
ical composition of the air, lessen the discomfort of the vitiated 
air as compared with the pigeon in jar A? 
4. From this experiment what would you conclude concerning 
the cause of the unpleasant effects of the air of an overcrowded 
and poorly ventilated room ? 90 
Laboratory Exercises in Physiology. 
50. Effect of an Artificial Atmosphere Containing an Excess 
of CO2 and a Deficiency of 0.,: A twenty-gallon gas tank is 
filled with an artificial atmosphere, in which the percentage of 
O2 is reduced by mixing with nitrogen and the CO, is increased 
by adding CO2 derived from the decomposition of marble by an 
acid, until the O2 and CO2 are present in the same proportion as 
in the last experiment (i. e., about 16 per cent of O2 and 3.5 to 
4 per cent of CO,). This is slowly drawn through a bell jar con- 
taining a pigeon. Observe that this pigeon does not show the 
same objective symptoms when breathing the artificial atmos- 
phere from a chemical source as the pigeon in jar A in the last 
experiment, which breathed air vitiated by respiration. Describe 
the result. 
51. Effect of Confining an Animal in a Small Space and in 
a Relatively Large Space: (1) One pigeon is placed in a 
small closed jar with a capacity of two liters, and kept there 
until distinct symptoms manifest themselves. Observe that, in 
addition to hypernea and dyspnea, the pigeon is restless, the 
pupils are dilated, and there is a condition of excitement and 
activity. (These results appear to be due to the deficiency of O2, 
as all the available O2 of the air of the jar is used up.) 
(2) Another pigeon is placed in a large bell jar with a capac- 
ity of ten liters and kept there five times as long as the first 
pigeon. Although the time of confinement is in the same pro- 
tion as the space, observe that the objective symptoms of the 
second pigeon are quite different from those of the first one. In 
addition to dyspnea there is dullness, drowsiness, and ultimately 
there would be coma, but no excitement or activity. The second 
pigeon seems to suffer more from the gradual accumulation of 
CO,, as there is still sufficient 0, present to support life. 
52. Tolerance to Foul Air (Bernard's Experiment): A 
small space containing a pigeon is placed in communication with 
the large bell jar used in the last experiment and the air of the 
two jars thoroughly mixed. 
Observe that the second pigeon, coming suddenly into an at- 
mosphere greatly vitiated by respiration, seems to suffer greater 
dyspnea and discomfort than the first one. Is there a tolerance 
to foul air? 92 
Laboratory Exercises in Physiology. 
Do the occupants of an overcrowded and poorly ventilated 
room notice the respiratory impurities, and stiffer as much dis- 
comfort as those who suddenly come into such an atmosphere 
from the pure air? 
53. Breathing Artificial Atmospheres: 1. The percentage 
of O2 is reduced by diluting air with nitrogen in a gas tank. 
The artificial atmosphere is then slowly drawn through a bell 
jar containing a pigeon. 
(a) Observe that an atmosphere containing approximately 
15 vol. per cent of 0, does not produce any objective symptoms 
or evidence of discomfort. 
(b) An atmosphere containing approximately 10 vol. per 
cent of O2 causes distinct hypernea and dyspnea. 
2. An artificial atmosphere containing an excess of CO2 is 
prepared by adding CO2, derived from the decomposition of 
broken marble by a mineral acid, to the air in different pro- 
portions. 
(a) Observe that an atmosphere containing as much as 2 per 
cent of CO2, derived from, a chemical source, does not cause 
symptoms or evidence of discomfort. 
(b) An atmosphere containing from 3 to 5 per cent CO, may 
not cause more than hypernea and dyspnea, if the proportion of 
CO, has been increased gradually. If an animal is suddenly 
placed in an atmosphere containing 5 vol. per cent of CO, there 
may be struggling, and at times syncope and unconsciousness, 
in addition to the disturbance of respiration. 
More than 5 per cent of CO, is distinctly dangerous, as the 
elimination of CO, from the blood is seriously interfered with, 
and may prove fatal. If the proportion of CO, is increased very 
gradually an animal may live for a short time in an atmosphere 
containing as much as 10 per cent by vol. of CO2. 
(c) Compare these proportions with the vitiation of the air 
by respiration. What degree of vitiation, as indicated by the 
volume per cent of CO,, is sufficient to produce distinct sub- 
jective symptoms from "crowd poison'' in a crowded room? 
What is the permissible limit of respiratory impurities for good 
ventilation ? 
54. Blood Gases: Gases will be extracted from the blood 
by a mercury pump. After exhaustion of the blood bulbs, they 94 
Laboratory Exercises in Physiology. 
are weighed and blood is drawn into them directly from the 
artery and from the vein of a dog. Some blood serum is placed 
in another blood bulb. The amount of blood is determined by 
weight and the volume calculated from that. 
The gases are collected in eudiometers and analyzed by absorb- 
ing the CO2 with caustic soda and the 0, with pyrogallic acid. 
Correction for temperature and pressure is made. 
1. Record the volume of arterial blood taken and the volume 
per cent of oxygen, CO,, and nitrogen, respectively. 
2. Record the volume of venous blood and the volume per 
cent of each gas extracted from it. 
3. Record the volume of blood serum and the volume per cent 
of gases extracted from it. 
4. Observe that a solution of NaIICO3 in distilled water does 
not give up its CO2, when exposed to a vacuum, as readily as 
blood serum. Explain the difference. 
From what you have seen, write a brief summary of the dif- 
ference in the proportion of the gases of arterial blood, venous 
blood and blood serum. How do O2 and CO, exist in the blood? 
55. The Volume of the Air Respired as Measured by the 
Spirometer: 1. The Tidal Air: The amount of air expelled 
from the lungs by each expiration in normal, quiet expiration is 
measured by breathing into the spirometer. The average of 
several observations is taken. This is usually 25 to 30 cu. inches 
or 400 to 500 c.c. 
2. The Supplemental or Reserve Air: This is the amount 
that can be exhaled by a forced expiration, after the tidal air 
has been expelled by natural expiration. The average amount 
is 90 to 100 cu. in. or 1500 to 1600 c.c. 
3. The Complemental Air: This is the greatest amount that 
can be taken into the chest by the greatest inspiratory effort, 
after the tidal air has been drawn into the lungs. It may be 
determined directly by filling the spirometer with air and meas- 
uring the amount that can be inspired after a natural inspira- 
tion. It varies in different individuals, usually being from 100 
to 120 cu. in. or 1600 c.c. to 2000 c.c. 
4. The Residual Air is the amount that remains in the lungs 96 
Laboratory Exercises in Physiology. 
after the greatest expiration. It is estimated to be 50 to 60 cu. 
in. or 800 to 1000 c.c. 
What proportion of the air in the lungs is replaced by fresh 
air at each respiration, i. e., what is the co-efficient of ventilation 
of the lungs or the proportion between the tidal air and the 
stationary air (residual plus the reserve air) ? 
56. The Vital or Respiratory Capacity: This is the sum of 
the complemental, tidal and supplemental or reserve air. It is 
the greatest amount that can be expired after the deepest inspira- 
tion and is easily measured with the spirometer. It usually 
amounts to 225 to 240 cu. in. or 3700 c.c. to 4000 c.c. in men, 
but is less in women. It is greatest in tall people and in those 
accustomed to physical exercise. 
Record your own vital capacity, and from the observations on 
different members of the class, write a brief summary of the 
influence of stature, body-weight, sex and active physical exer- 
cise upon the respiratory capacity. 
57. Chest Expansion: With a tape measure take the girth 
of the chest at the level of the fifth or sixth rib (a) after the 
greatest expiratory effort and (b) after the deepest inspiration. 
The difference between the two gives the greatest chest expansion. 
It varies from 2.5 to 4.5 inches in different individuals. Women 
have relatively a greater chest expansion than men, as the result 
of thoracic breathing, although the respiratory or vital capacity 
is less in women than in men. 
From the observations upon different members of the class, 
note that there is no constant relationship between the chest ex- 
pansion and the respiratory or vital capacity. Describe the in- 
fluence of stature and of physical exercise upon the chest ex- 
pansion. 
58. Intrathoracic and Intratracheal Pressure: A cannula is 
introduced through the intercostal muscles of an etherized dog 
into the pleural cavity and connected with a water manometer 
to measure the intrathoracic pressure. Another cannula is 
pushed into the trachea between the cartilaginous rings and con- 
nected with another water manometer to register the pressure 
within the trachea. 
1. Observe that the intrathoracic pressure is always negative, 98 
Laboratory Exercises in Physiology. 
even at the end of natural expiration, and that the negativity 
increases with each inspiration. Record the average readings of 
the manometer at the end of expiration and during inspiration. 
Describe the cause of (a) the negative intrathoracic pressure 
when the chest is at rest, and (b) of the change with each in- 
spiration. 
2. Observe that the intratracheal pressure is negative during 
inspiration and positive during expiration. (This is also true of 
the intrapulmonary pressure.) Record the registration of the 
manometer and explain the cause of the changes of pressure in 
the different phases of respiration. 
3. Observe the effects of obstructing the upper air passages 
(by pressing the ether cone tightly over the nose) upon the intra- 
tracheal and the intrathoracic pressure. Record the results. 
4. Observe the pressure within the trachea and within the 
thorax after the trachea is opened. Record the result. 
5. When the tracheal tube is obstructed by placing the finger 
over it, to what extent may the intratracheal and intrathoracic 
pressure be varied by the labored respiratory efforts? Record 
the results and write a brief account of the mechanism of 
natural respiration. 
59. Graphic Record of Respiratory Movements and Relation 
to Respiratory Sounds: The stethometer may be used to show 
the changes in the different diameters of the chest with respira- 
tion, but the stethograph or pneumograph is the most convenient 
means of recording the respiratory movements in man. This is 
essentially a receiving tambour. It is fastened tightly about the 
chest, so that the movements of the chest wall in inspiration and 
expiration are easily recorded on a moving drum by means of 
a recording tambour. An electro-magnet is brought to write 
directly under the recording tambour. This is connected to a 
couple of dry cells with a simple key for marking the time of the 
respiratory sounds as one auscults the chest. 
The drum is set at slow speed. One person auscults the chest 
and with one hand on the key marks the beginning and end of 
the vesicular sound by making and breaking the current. Care 
should be taken to get a tracing of natural, quiet respiration as 
the normal. Take a time tracing of seconds or fifths of seconds 
with the electro-magnet. 100 
Laboratory Exercises in Physiology. 
1. Observe that the expansion of the chest, produced by 
active muscular contraction during the phase of inspiration, is 
shorter than the passive return of the chest wall during the 
phase of expiration. 
2. Observe that there is no pause between inspiration and 
expiration, but there may be a pause after each expiration if 
the respirations are slow. 
3. Observe that the vesicular sound begins early in the phase 
of inspiration and continues into the phase of expiration. The 
inspiratory portion of the vesicular sound is much longer than 
the part that occurs in the phase of expiration. The expiratory 
part of the vesicular sound only occupies the first third of the 
phase of expiration. 
Write a brief statement of your observation as to the relative 
time occupied by the respiratory movement and the respiratory 
sounds. 
60. Forced Breathing and True Apnea: (a) Take a trac- 
ing of normal respiration. The subject then breathes 18 times 
per minute, taking care to draw deep inspirations but not to 
make^ active expirations. After 30 or 40 seconds the voluntary 
effort is discontinued and the respiration is allowed to occur in- 
voluntarily. In typical cases of apnea the respiration ceases 
altogether for 10-30 seconds, or the respirations become very 
shallow. This is due to the decreased CO2 in the blood (acapnea) 
from the deep forced respirations. As soon as it accumulates up 
to the normal the respiratory center is stimulated to restore the 
usual depth and rate of respirations. 
If the subject breathes too rapidly, or if the muscular exer- 
tions are too vigorous, especially in expiration, the production 
of CO2 may be increased to such an extent that the CO2 of the 
blood is not decreased, and apnea does not occur. 
(b) Prolonged Forced Breathing for two or three minutes 
causes a longer period of apnea toward the end of which slight 
cyanosis of the lips may occur from the want of oxygen. This 
may cause irregular respiration for several minutes after the 
period of apnea. Prolonged forced breathing may cause "light- 
headedness", shivering, and muscular weakness. 
(c) Forced Breathing Without Excessive Elimination of 
CO2: A large paper bag is held tightly over the nose and mouth 102 
Laboratory Exercises in Physiology. 
while the subject does forced breathing, as before. The peculiar 
and disagreeable sensations of acapnea do not occur, as rebreath- 
ing the same air prevents excessive loss of CO2. The voluntary 
effort is less than in the previous experiments. The subject does 
not experience apnea and may even be unable to stop breathing 
voluntarily. This shows that the apnea in (a) and (b) was due 
to an alteration in the blood gases and not to a nervous reflex 
(vagal apnea). 
61. Voluntary Apnea. 
1. The subject holds his breath after a moderate (normal) 
inspiration. The breaking point usually occurs after about 40 
seconds. 
2. The breath is held after a very deep inspiration. A 
longer time elapses (usually 50 or 60 seconds) before the break- 
ing point is reached. 
3. The subject performs forced breathing for a couple of 
minutes. The breath can then be held for two or three minutes. 
4. The subject performs forced breathing for a couple of min- 
utes, but this time in the paper bag. Note that the breath can- 
not be held as long as before. 
Take tracings of all the above. Take time tracings in seconds 
in every experiment. 
62. Asphyxia: When the entrance of air into the lungs is 
prevented in any way, or when the blood is not oxygenated, the 
following stages of asphyxia appear: 
1. A stage of hypernea lasting about one minute. The res- 
pirations are increased in frequency and in depth. The inspira- 
tory phase of the excited respiratory movements is especially 
prominent. Great variations of the blood-pressure occur with 
the efforts at respiration and the heart beats rapidly. 
2. A stage of dyspnea and convulsions. Respiration is la- 
bored and the movements become convulsive. During the sec- 
ond stage the expiratory movements become more prominent. 
There is extreme cyanosis; the pupils are widely dilated; the 
struggling movements pass into irregular spasms from stimula- 
tion of the nerve centers. The second stage lasts about one 
minute. 
Cardiac inhibition is present from stimulation of the cardio- 104 
Laboratory Exercises in Physiology. 
inhibitory center by the venosity of the blood; the arterial 
pressure usually rises, in spite of the cardiac inhibition, from 
stimulation of the vasoconstrictor center. When general con- 
vulsions occur these contribute to the rise of pressure. 
3. The third stage is one of collapse which follows the irregu- 
lar spasm of the second stage. Consciousness and reflex activity 
are lost and there is a condition of complete relaxation. Res- 
piratory movements occur at long and irregular intervals, in 
which violent inspiratory efforts are made. The blood-pressure 
is low as the result of paralysis of the vasomoter center. The 
heart continues to beat for a considerable time after the respira- 
tory movements cease. For this reason prompt efforts at re- 
suscitation may be successful. 
The third stage lasts two or three minutes or longer. In 
adults recovery does not occur after more than four or five 
minutes of complete asphyxia or suffocation, but young animals 
may recover after a longer period. In resuscitating a person 
or an animal it may be necessary to keep up artificial respiration 
for twenty to thirty minutes, or even longer, before spontaneous 
respirations occur. 
63. Artificial Respiration: During the third stage of as- 
phyxia, or after the respiratory movements cease from any 
cause, the head is lowered and the feet elevated; the mouth and 
pharynx are cleared; the tongue is polled forward rhythmically 
(Laborde's method) to stimulate the respiratory center reflexly, 
traction being made during the inspiratory phase of artificial 
respiration. 
In animals air is expelled from the lungs by pressure upon 
the ribs. These active respiratory movements alternate with the 
pulls made upon the tongue. The elasticity of the chest wall 
causes the thorax to expand and air to enter the lungs. 
In man the Sylvester method consists in expanding the chest 
(i. e., producing inspiration) by drawing the arms up over the 
head, the tongue being pulled forward. The expiratory move- 
ment is produced by pressing the patient's elbows against the 
sides of the chest. The combined Laborde-Sylvester method 
gives the best results. 
When artificial respiration must be carried on by one person it 
is best to place the patient face down and compress the thorax 106 
Laboratory Exercises in Physiology. 
by pressure applied postero-laterally, allowing the thoracic wall 
to expand by its own elasticity (Schafer). 
Write a brief description of the restoration of the circulation 
and respiration by maintaining artificial respiration, giving the 
time occupied. 
NERVOUS MECHANISM OF RESPIRATION. 
64. Motor or Efferent Nerves: The Phrenics: Observe the 
contractions of the diaphram from stimulation of one phrenic 
nerve. Note the effect of cutting one phrenic; of stimulating the 
peripheral end. 
Note the effect of cutting both phrenics. What change is there 
in the type of respiratory movements after section of both 
phrenics ? 
Recurrent or Inferior Laryngeal: Observe the condition of the 
vocal cord of the same aside during stimulation of this nerve. 
How does irritation or paralysis of this nerve change the voice? 
What is the the condition of the vocal cords in coughing? 
65. Afferent Nerves of Respiration: Apnea: In a normal 
animal, with all the nerves intact, observe the temporary cessation 
of the respiratory movements after rapid inflation of the lungs. 
Explain the cause of this apnea. 
Observe the effect upon the respiratory movements of stopping 
the respiration by occluding the tracheal cannula at the end of 
inspiration. Explain the result of distending the chest. 
In the same way observe the effect of suddenly stopping res- 
piration at the end of natural expiration. Explain the result. 
Respiratory Rate and Volume Before and After Section of the 
Vagi: The normal respiratory rate per minute is counted and 
recorded. The volume of air exhaled from the lungs for periods 
of two or three minutes is measured by the spirometer. An aver- 
age of several observations is taken and recorded. 
Later in the experiment, after both vagi have been severed, the 
respiratory rate and volume are again determined. Record the 
results. Observe that the volume is practically the same, al- 
though the rate is greatly reduced. Explain the cause of the 
slow respiration after the section of the vagi. 108 
Laboratory Exercises in Physiology. 
Stimulation of the Nasal Branches of the Trigeminal Nerve: 
Observe the inhibition of respiration from stimulation by blowing 
tobacco smoke or some irritating vapor into the nose of an ani- 
mal. What is the cause of the inhibition of respiration early in 
anesthesia? How may this condition be distinguished from the 
cessation of respiration caused by the depressant effect of the 
anesthetic upon the respiratory center? 
Superior Laryngeal Nerve: (a) What results from stimula- 
tion of this nerve with a weak current ? (b) With a strong cur- 
rent? In what phase is respiration inhibited? 
Sensory Nerves: Observe the increase in rate and depth (ac- 
celeration) of the respirations from stimulation of any mixed 
spinal nerve containing sensory fibers, as the central end of the 
cut sciatic. 
Respiratory Fibers of the Vagi: Note the effect of cutting (a) 
one vagus and (b) of cutting both vagi upon the respiratory rate. 
Observe the effect of stimulating the central ends of the se- 
vered vagi, beginning writh a very weak current and gradually 
increasing its strength. Record the result. What phase of 
respiration is especially affected? 
What is the effect of suddenly stimulating the central ends of 
the vagi with a strong current? Which phase is particularly 
affected? What kinds of respiratory fibers exist in the pneumo- 
gastrics ? 
Observe the result of occluding the tracheal tube at the end 
of inspiration and at the end of expiration, respectively, and 
compare with the result obtained by the same procedure before 
the vagi were cut. Explain the difference. 
Inflate the lungs rapidly and observe whether apnea can be 
produced as readily after section of both vagi as can be done 
when they are intact. Explain the result. 
From this experiment write a brief description of the respira- 
tory functions of the afferent fibers of the vagi from the lungs, 
telling how they may be demonstrated experimentally and how 
they are stimulated under normal conditions during natural res- 
piration. 110 
Laboratory Exercises in Physiology. 
LYMPH AND CHYLE 
A dog has been fed on fat meat several hours before the 
experiment. The animal is etherized; the thoracic duct is 
exposed just before it empties into the jugular vein and a can- 
nula is tied in it; another cannula is placed in the principal 
lymphatic trunk coming from the head. 
66. Appearance of Lymph and Chyle: Observe the milky 
chyle as it drops from the cannula in the thoracic duct, and 
compare it with the colorless lymph which comes from the 
lymphatic trunk of the head. Explain the difference. 
Study the appearance of a drop of chyle under the microscope 
and compare it (a) with a drop of milk and (b) with a soap 
emulsion made by shaking a rancid oil with a solution of sodium 
carbonate. Judged by the minute division and uniformity in 
the size of the fat globules, how does chyle compare with the 
other emulsions? How does the emulsification of fats in the 
chyle compare with that which occurs in the intestine during 
the digestion of fats? 
Observe the influence of inspiration and expiration, respec- 
tively, upon the flow of chyle. Explain the difference in the 
two phases of respiration. Count the number of drops of lymph 
and of chyle for periods of two minutes and record them in 
separate columns in a protocol of the experiment. Make two 
observations for the normal. 
67. Massage: Count the number of drops from the cervical 
lymphatic during massage of the corresponding side of the head 
and neck. Record the result of two observations. From this 
experiment what do you consider the greatest benefit of local 
massage? 
68. Lymphagogues: These are substances which cause an 
increase in the formation and flow of lymph when they are 
injected into the blood. A study of their action gives a better 
understanding of the formation of lymph and the conditions 
which influence it. 
There are two classes of lymphagogues: (1) Crystalloids, 
like dextrose, urea, sodium chloride, etc., which increase the 112 
Laboratory Exercises in Physiology. 
lymph by producing a condition of hydremic plethora in the 
blood and a consequent increase in the blood pressure in the 
capillaries. If the plethora be prevented by bleeding, the 
hydremia alone does not cause an increase in the formation of 
lymph. 
(2) Colloidal lymphagogues, like commercial peptone, egg 
albumin, extracts of leech heads, crayfish, mussels, etc., and 
curare, which seem to act by increasing the permeability of 
the vessel walls. With the exception of curare, they appear 
to act chiefly upon the blood capillaries of the liver, while the 
crystalloid lymphagogues act uniformly upon all the vessels 
of the body. 
A definite amount of blood is taken from the dog. Observe 
that the flow of lymph and chyle is ditninished as a result. 
Clotting of the blood is prevented by receiving it directly 
into a 2.5 per cent solution of sodium citrate in the proportion of 
10:1, so that a mixture of 0.25 per cent citrated blood is obtained. 
This is returned to the veins and the flow of lymph and chyle 
is determined by counting the drops for two-minute periods 
as before. The small amount of sodium citrate used to prevent 
coagulation of the blood causes very little, if any, increase in 
the flow of lymph. 
The same amount of blood is withdrawn a second time and 
mixed with the citrate solution in the same proportion. 
Sufficient dextrose is then injected intravenously to abstract 
from the tissues, by its osmotic pressure, an amount of fluid 
equivalent to the quantity of blood removed by bleeding. 
Observe that the flow of chyle is not increased, or if so only 
slightly, although the blood has been made hydremic by the 
water attracted from the tissues by the dextrose. Record the 
number of drops for two-minute periods as before. 
After waiting five or ten minutes, the citrated blood will be 
injected into the vein at the temperature of the body. The flow 
of lymph may be increased for a short time. As soon as it 
returns to the normal, dextrose is again injected in exactly 
the same quantity as before. Observe the great increase in 
the flow of lymph from the lymphatic from the head, and of 
chyle from the thoracic duct. Count the number of drops 
again for two minute periods and record the results. 114 
Laboratory Exercises in Physiology. 
In this case there is a sudden plethora in addition to the 
hydremia. Crystalloid lymphagogues seem to act upon all the 
vessels of the body by physical forces to a large extent, the 
increase in the capillary blood pressure causing an increase in 
the formation of lymph. This is not the sole cause of the 
increased lymph flow, since it lasts longer than the increase 
of venous pressure. When the crystalloid lymphagogues are 
injected shortly before the death of an animal, the increased 
lymph flow continues long after the death of the animal and 
may reach its maximum a considerable time after the capillary 
pressure ceased to exist. 
After the flow of lymph and chyle has subsided to the normal, 
a solution of commercial peptone (0.1 gram Witte's peptone 
per kilo of body weight) is injected intravenously. Observe 
that the flow of lymph from the head is not changed, but the 
flow of chyle from the thoracic duct is greatly increased. Count 
the number of drops for two-minute periods and record the 
result as before in a protocol of the experiment. 
It has been found by experiment that the increased flow of 
lymph from the thoracic duct after the injection of peptone 
comes from the liver; also that the lymph is more concentrated 
and contains a higher proportion of solids in solution, whereas 
the lymph formed after the injection of crystalloids is more 
dilute and watery than the normal. 
These facts indicate that colloidal lymphagogues do not act 
by known physical laws, but that the endothelial cells lining 
thb blood capillaries of the liver become more permeable, prob- 
ably through some change in their vital properties. 
69. Lacteals: At the end of the experiment, when the ab- 
domen of the dog is opened, observe the lymphatic vessels, or 
lacteals, of the intestinal wall and mesentery; observe their 
relation to the lymph glands of the mesentery. 
70. Absorption of Crystalloids and of Colloids from the 
Tissues: A dog is etherized; through a lumbar incision the 
ureter is drawn out and a cannula tied in it so that the urine 
may be collected as it comes directly from the kidney. The 
normal urine is first collected and examined to be certain that 
albuminuria does not exist. 116 
Laboratory Exercises in Physiology. 
A solution of egg albumin (as a type of a colloid) is injected 
subcutaneously into one thigh; a solution of potassium ferro- 
cyanide (as a type of crystalloid) is injected into the other 
thigh. Both of these substances are eliminated by the kidneys 
within two minutes when they are injected into the blood. 
The presence of potassium ferro-cyanide may be detected by 
the Prussian blue test; egg albumin may be recognized by 
heating and acidulating the diluted urine or by the ring test 
with HNO3. 
Note the exact time at which the injection is made and make 
a protocol of the experiment. Observe that the crystalloid 
appears in the urine in a few minutes (usually about 8 or 10 
minutes), while the colloid (egg albumin) can not be detected 
in the urine within an hour or more after it has been injected 
hypodermically. 
The marked difference in the time required for absorption 
suggests that crystalloids are absorbed by direct diffusion from 
the lymph spaces through the thin capillary walls into the 
blood. Alkaloids are also probably absorbed in that way. 
Colloids are probably taken up from the lymph spaces with 
the lymph and carried by the sluggish lymph stream until they 
reach the blood through the thoracic duct. 
The difference in absorption of colloids and of crystalloids 
may also be shown by injecting a solution of carmine, as a 
type of a relatively non-diffusible pigment, and methyl blue 
as a diffusible substance. The color of these substances enables 
them to be recognized readily, both in the lymph and in the 
urine. 
71. Absorption of Foreign Particles from the Large Serous 
Cavities: An emulsion of a finely divided insoluble pigment 
(water color lampblack in physiological salt sol.) is injected 
into the peritoneal cavities of two rabbits. One rabbit is killed 
two hours after the injection and the other after twenty-four 
hours. 
(a) Observe that the lampblack collects in lines on the 
omentum and on the diaphragm and that it can not be washed 
off except by using violence. Note that the mediastinal lymph- 
atic glands are colored black by the pigment granules, even two 
hours after the injection. 118 
Laboratory Exercises in Physiology. 
(b) Observe that at the end of twenty-four hours there is 
no free pigment in the abdominal cavity. 
(c) Study microscopic specimens of the omentum from rab- 
bits after the injection of lampblack. Note that the pigment 
particles are held on the surface of the omentum by a fibrinous 
exudate; after twenty-four hours the pigment granules are 
found in the large cells ("trailers") of the omentum. 
(d) Examine microscopic sections through the mediastinal 
lymph glands of rabbits killed two and twenty-four hours, 
respectively, after the intraperitoneal injection of lampblack. 
Observe the distribution of the pigment granules in each and 
describe the appearance of the section of each gland. 
This experiment has an important practical bearing upon the 
phenomena of absorption in cases of peritonitis and also upon 
the function of the omentum. The fact that the omentum picks 
up foreign particles and holds them in its meshes and cells, 
shows that it has a protective function in localizing infections 
similar to that of the lymphatic nodes. 
The rapidity with which foreign particles pass out of the 
abdominal cavity into the thoracic lymph glands explains the 
widespread general disturbances which occur so soon when 
infectious material escapes into the peritoneal cavity, as from 
perforation of the intestine, vermiform appendix, etc. 120 
Laboratory Exercises in Physiology. 
THE CIRCULATION. 
THE HEART. 
72. Arrangement of the Muscle Fibers of the Walls of the 
Heart: A beef's heart has been boiled for two hours to loosen 
the muscle fibers by changing the connective tissue to gelatine 
and dissolving it. In the thin auricular walls it is difficult 
to separate the layers, but there is an external transverse and 
an internal longitudinal layer. Observe the, way in which the 
veins open into the right auricle and the great abundance of 
muscle fibers surrounding the mouths of these veins. 
In the walls of the ventricles three distinct layers may be 
made out: (a) An external spiral or oblique layer; (b) a middle 
transverse or circular layer; and (c) an inner longitudinal 
layer. Tear the fibers of the thin external layer and note the 
spiral arrangement, downward and to the left; observe the 
"figure of eight" turn by which they become continuous with 
the internal layer and form a whorl at the apex. 
Note that the middle transverse layer of fibres is much 
thicker than the other two, especially at the base of the ven- 
tricles. This is a matter of extreme importance because it 
serves to lessen the size of the auriculo-ventricular orifies dur- 
ing systole, when the valves are closed, thereby rendering them 
competent. Degeneration of the heart muscle may result in 
incompetence of these valves without any disease of the valves 
themselves. 
Observe that the inner longitudinal layer extends into the 
muscular columns and papillary muscles of the ventricular wall. 
Compare the relative thickness of the walls of the two ven- 
tricles. Record the result of this observation and explain why 
the wall of the left' ventricle is so much thicker than that 
of the right side. 
73. The Auriculo-Ventricular Bundle or the Bundle of His: 
This can readily be seen in the heart of the sheep, calf or ox. 
In the heart of other mammals this bundle can not be seen 
readily by the unaided eye, but it may be traced by histological 
examination. Examine microscopic sections of this bundle. 122 
Laboratory Exercises in Physiology. 
Observe the origin of the auriculo-ventricular bundle beneath 
the endocardium of the right auricle; trace it benealh the tri- 
cuspid valve into the ventricular septum and observe that it 
divides into two parts, one of which is distributed on the 
inner surface of each ventricle. 
This bundle consists of special muscle fibres which conduct 
the contraction wave and form a continuity of structure be- 
tween the auricles and ventricles. It was formerly thought 
that the fibrous auriculo-ventricular ring completely separated 
the muscular wall of the auricles from that of the ventricles 
in the mammalian heart. 
74. Valves and Orifices of the Heart: Observe the arrange- 
ment of the two sets of auriculo-ventricular valves, and the 
semi-lunar valves; the direction in which they open, their size, 
attachments, etc. 
Compare the size of the orifices. 
Observe the origin and distribution of the coronary arteries. 
75. Change in Shape of the Heart During Action: This is 
shown by hearts that have been arrested and fixed while in 
systole and in diastole. Note (1) that in systole the antero- 
posterior and transverse diameters are shortened, and that the 
long axis of the heart is also somewhat shortened, though to a 
much less extent. Observe (2) that during systole the apex 
becomes more pointed and that the general outline of the 
heart is decidedly cone-shaped; (3) that in diastole the gen- 
eral contour of the heart is more globular and the apex less 
pointed; (4) that in diastole the transverse diameter is the long- 
est; (5) viewed from the base, the contour of the heart in 
diastole is elliptical, while in systole it is circular. 
76. The Valves of the Heart in Action. Gad's Heart 
Windows: A glass window with a side tube has been tied 
in the pulmonary artery of a fresh calf's heart above.the semi- 
lunar valves; another window has been placed in the right 
auricle, so as to look down on the tricuspid valve from above. 
A rubber bulb is tied into the apex of the right ventricle to rep- 
resent systole of the heart when it is pressed upon; a small 
electric lamp illuminates the interior of the ventricle. The fluid 
enters the right auricle from a pressure bottle and is pumped 124 
Laboratory Exercises in Physiology. 
out of the ventricle into the pulmonary artery against a column 
of fluid, which closes the semilunar valves during diastole; a 
tube carries the fluid from the pulmonary artery into a funnel 
in the pressure bottle. A bandage is tied tightly about the 
base of the ventricles to lessen the size of the tricuspid orifice 
and prevent regurgitation into the auricle. Note (a) that when 
the bulb is compressed (i. e., in systole) the edges of the 
tricuspid valve come together, closing the orifice, thus pre- 
venting the fluid from passing back into the auricle, and com- 
pelling it to pass out through the pulmonary artery; that they 
open as soon as the bulb is relaxed (diastole), (b) Observe the 
shape and disposition of the leaflets of the valve, and the action 
of the chordae tendineae. (c) Note that the semilunar valves 
open in systole and close in diastole (i. e., when the auriculo- 
ventricular valves are open, the semilunar valves are closed, 
and vice versa). 
Why is the tricuspid valve incompetent in the dead heart, 
unless a bandage is wrapped about the base of the ventricle? 
What is the function of the valves of the heart? 
77. Verdin's Model of the Heart Valves: This is a me- 
chanical device for showing the action of the heart valves in 
determining the direction of the current through the heart. 
Note that only one valve is open at a time (i. e., when one is 
open the other is closed). 
78. The Competence of the Semilunar Valves: The pulmo- 
nary artery has been cut away from the right ventricle of a 
calf's heart, and connected with an upright tube. Note how 
well the leaflets of the semilunar valve close off the opening 
when the tube, reaching to a considerable height, is filled with 
wTater so as to exert pressure on them from above. 
79. To Expose the Frog's Heart: Pith the frog by intro- 
ducing a hat pin between the skull and atlas and moving it 
laterally. This severs the connection between the brain and 
spinal cord, and destroys all conscious sensation. Pin the frog 
out on the frog plate, ventral surface up, with U-shaped tacks. 
With stout scissors make a median ventral incision through the 
skin from pelvis to jaw; cut through the abdominal wall, 
avoiding the epigastric vein which runs in the median line. 126 
Laboratory Exercises in Physiology. 
Tie this vein near the pelvis. Continue the incision upward, 
cutting through the bony pectoral girdle and taking care not 
to injure the parts beneath. Pin back the flaps. Lift up the 
pericardium with the forceps and cut it open. 
80. General Arrangement of the Frog's Heart: The frenum 
(a delicate band of connective tissue containing a vein) runs from 
the pericardium to the posterior wall of the ventricle. With a 
curved metal "seeker," pass a ligature, moistened with physiolog- 
ical salt solution(0.6 percent NaCl)about this and cut it posterior 
to the ligature. Anteriorly, observe the two auricles above; the 
single ventricle forming the apex below; the two auricles being 
separated from the ventricle by a distinct groove, the auriculo- 
ventricular groove. Coming off from the ventricle anteriorly, and 
projecting in front of the auricles, is the bulbils arteriosus which 
divides into two aortae. Each of these divides into three vessels. 
With the ligature attached to the frenum, tilt up the heart and 
observe the sinus venosus which is continuous with the right 
auricle; it receives the blood from the single large inferior vena 
cava and the two superior venae cavae. The sinus, auricles 
and ventricle can be seen with the apex titled up; the auricles, 
ventricle and bulbus arteriosus can be seen with the heart in 
its normal position. 
81. The Heart in Action; The Cardiac Cycle: Observe (1) 
that the contraction (systole) begins in the sinus venosus, and 
rapidly extends in regular order to the auricles and ventricle; 
(2) that the period of activity (systole) in each part of the 
heart is followed by a longer period of rest or diastole. If 
the heart is beating too rapidly to make out the sequence of 
events, apply cold physiological salt solution with a pipette until 
the heart beats slowly. Cold slows the heart and enables one 
to study the cycle of events. When the heart beats slowly the 
entire heart may be in a condition of diastole between the 
periods of activity; (3) note the difference between the dias- 
tole of the entire heart and the diastole of the individual chambers 
of the heart; (4) does the systole of the auricles occupy as long 
a time as the systole of the ventricle? 
Observe the pronounced change of color of the ventricle as it 
passes from diastole into systole, while there is comparatively 128 
Laboratory Exercises in Physiology. 
little change in the color of the auricles. The ventricle seems 
to empty itself completely with each systole while the auricles 
appear to contain some residual hlood. 
The frog's heart does not have coronary blood vessels, like a 
mammalian heart, but is nourished by the blood in its cham- 
bers. The blood spaces of the spongy ventricular wall are filled 
from openings on its inner surface during diastole and emptied 
during systole so that the change of color is much greater than 
in the mammal. 
What alteration in the shape of the ventricle occurs during 
systole and diastole? (The frog's heart shortens in its long axis 
much more than the mammalian heart.) 
What change in the position of the apex of the heart and in 
the position of the auriculo-ventrieular groove, or base of the 
heart, takes place with each systole? 
Graphic Record of the Heart Beats by the Gaskell Lever: 
Lift the heart by grasping the tip of the ventricle with fine 
forceps and tie a silk ligature as close to the forceps as possible, 
so that only a little of the ventricular wall at the apex is in- 
cluded in the ligature. By pulling on the ends of the ligature 
gently, so that the thread just fails to slip between the thumb 
and finger, it will sink in the spongy tissue of the heart suffi- 
ciently to hold without cutting through. 
The long ends of the ligature are passed through a short 
piece of rubber tubing of large lumen for the next experiment 
and are then tied to the short arm of a Gaskell lever. Adjust 
the lever and frog-plate so that the ligature pulls in a vertical 
line, causing the long arm of the lever to write lightly upon 
the drum. The short arm of the Gaskell lever may be counter- 
poised. 
Connect the electro-magnet with the time-marking current 
arranged to mark seconds, and bring it to write under the 
Gaskell lever. Take a tracing with the drum at moderate or 
slow speed. 
82. Influence of Temperature Upon the Heart Beats: With 
everything adjusted as in the last paragraph, take a short 
tracing (four or five inches) of the normal heart beat, and 
record under it the room temperature. (The temperature of 
the frog is the same as that of the air.) Cool some Ringer's 130 
Laboratory Exercises in Physiology. 
sol. by placing a beaker of it in a pan containing cracked ice 
and salt. With a pipette apply the cold .Ringer's sol. keeping 
the rubber tubing filled so that the heart is completely im- 
mersed for two or three minutes, or longer if necessary, until 
the rate of the heart is slowed. Continue to apply the cold 
Ringer's sol. during the tracing, taking care not to interfere 
with the writing of the lever. Take tracings "when the temper- 
ature has been reduced to 15° C.; also at 10° C., 5° C. and 1° 
C., respectively, with the drum at the same speed each time. 
Now warm some Ringer's sol. over a flame and when the 
temperature reaches 30° to 32° C. apply it to the heart with 
a pipette as before. After applying it for several minutes, 
take a tracing as before, with the drum at the same speed 
and the electro-magnet registering seconds. Heat the physio- 
logical salt sol. to 37° C. and apply it to the heart until the 
rate shows acceleration. Be careful that the temperature does 
not go above 38° C., as a high temperature causes the heart 
to go into heat rigor and lose its vital properties. 
What are the effects of heat and cold, respectively, upon the 
rate of the heart beat? Is the height of the contraction waves 
changed ? 
Caution: Apply the sol. long enough to change the rate each 
time before starting the tracing on the drum and continue to 
let it run while the tracing is taken. 
83. The Excised Heart: Remove the heart lever and rub- 
ber tubing. Count the number of heart beats per minute. Lift 
the heart by the ligature attached to the apex and carefully 
excise it, including the sinus venosus, by cutting the vessels as 
far away from the heart as possible. Place the heart in a watch 
crystal and again count the rate, keeping the heart moistened 
with Ringer's sol. 
(a) Has removal of the heart from the body changed its 
rate or rhythm ? 
(b) Has the sequence of events in the heart been changed? 
(c) Does the activity of the heart depend upon nerve im- 
pulses from the central nervous system? 
(d) Is the circulation of blood through the frog's heart 
necessary for its action? 
Now count the beats of the heart again and record the rate 132 
Laboratory Exercises in Physiology. 
at the room temperature. With a pipette apply cold Ringer's 
sol. (at the temperature of 15° and 10° C., respectively) for 
several minutes and again count the rate. Then apply warm 
Ringer's sol. (not over 38° C.) for several minutes and count 
the rate. Record the number in each ease. Is the excised 
heart influenced by heat and cold in the same way as the normal 
heart when it is connected with the nervous system? 
Float the heart in Ringer's solution in the watch crystal; 
with delicate forceps pick up the sinus venosus and with fine 
scissors cut it off. 
(a) Does the sinus continue to beat? 
(b) Does the rest of the heart beat? 
(c) Is the rhythm the same in the two parts? 
Now sever the auricles from the ventricle by cutting through, 
or just below, the auriculo-ventricular groove. 
(d) Do the auricles and ventricle continue to beat? 
(e) If the ventricle does not beat is it capable of beating? 
(Determine this by pricking it with a needle or sharp forceps.) 
Observe that there is a single contraction for each stimulus, 
and not a series of contractions. 
PHYSIOLOGICAL PECULIARITIES OF HEART MUSCLE. 
Note.-Before performing the experiments of the next exercise, the 
following phenomena in voluntary muscle will be demonstrated to the 
class: 1. Submaximal contractions from weak electrical stimuli. 
2. The time of the single contraction or twitch (0.1 second). 3. 
The very short latent period (0.01 second), which is also the re- 
fractory period in voluntary muscle. 4. Tetanus caused by a rapid 
succession of stimuli, which cause a fusion of separate contractions. 
5. Contractions caused by the galvanic current at its make and 
break. 
84. Extra Contractions and Refractory Period: Pith a frog 
so that it can not feel any pain. Expose the heart; tie the 
frenum and pin the frog on the cork plate. Tie a silk ligature 
to the apex of the heart and then tie the free ends over the 
short arm of a Gaskell lever as in the last experiment. 
Arrange the inductorium for single induction shocks with a 
simply key in the primary circuit. To the binding posts of the 
secondary coil fasten the wires of special electrodes imbedded 134 
Laboratory Exercises in Physiology. 
in cork. By means of pins in the cork, arrange the electrodes 
so that both of the looped, platinum tips are constantly in 
contact with the heart muscle without interfering with its 
contractions. 
Place the electro-magnet in the primary circuit by shunting 
from the binding posts of the primary coil for single induction 
shocks, and bring it to write on exactly the same vertical 
line as the heart-lever. This will mark the point of stimulation. 
Adjust the secondary coil to such position that induction 
shocks are felt by the heart both at the make and break of 
the primary circuit. Set the drum at moderately low speed. 
If the heart is beating rapidly, slow it by applying cold 
physiological salt solution with a pipette. Take a tracing of 
normal beats (four or five inches on the drum). Now, with the 
drum at the same speed, stimulate with singly induction shocks 
of sufficient strength to be felt by the heart. Try to apply 
these stimuli at different periods of the cycle of the heart's 
action and gradually increase the frequency of stimulation, 
taking care that the electro-magnet registers the point of stim- 
ulation exactly under the heart-lever. 
Do all of the stimuli cause extra contractions? If not, ex- 
plain why some fail to do so. What is the refractory period 
in the heart? Observe that after a number of extra contrac- 
tions occur in rapid succession from frequent stimuli, there is a 
"compensatory pause" before the heart returns to its regular 
rhythm. This momentary slowing of the heart offsets the un- 
usual activity from artificial stimulation. Before the paper 
is taken from the drum the electro-magnet should be connected 
with the time-marking current for recording seconds under 
the tracings with the drum at the same speed as above. 
85. Inhibition of the Heart by Stannius' Ligature: With a 
curved seeker clear the two aortae from the auricles and pass 
a silk thread, moistened with physiological salt sol., between the 
aortae and superior venae carvae. With the ligature attached 
to the apex turn it up toward the head, and observe the 
"crescent," i. e., a V-shaped or crescentric white line, caused 
by a collection of nerve cells, which occurs just at the junction 
of the sinus venosus with the right auricle. Tie the silk liga- 
ture just over this crescent. This is the "first ligature of 136 
Laboratory Exercises in Physiology. 
Stannius." Observe that the sinus continues to beat in its 
normal rhythm while the auricles and ventricle stop in the 
phase of diastole and remain quiescent for a variable time. 
(The effect of this ligature will be explained more fully later, 
under the heading of the nervous control of the heart.) 
Note.-If the ventricle starts to beat spontaneously during the 
following experiments, tie another ligature to the auricular side of 
the first ligature of Stannius described above. 
86. Staircase Contractions: Return the heart to its normal 
position and fasten the ligature attached to the apex to the 
.short arm of the Gaskell lever as before. Arrange the electrodes 
so that both platinum wires are in contact with the heart 
muscles as before. Bring the electromagnet, shunted from the 
primary circuit, to write exactly in the same vertical line with 
the Gaskell lever. 
Starting with the secondary coil at right angles to the pri- 
mary, stimulate with single induction shocks, increasing the 
strength of the current until a contraction is obtained at the 
break of the current. Set the drum at slow speed. Now re- 
peat this minimal stimulus at intervals of one second or more, 
while the drum is moving at slow speed. Are all the contrac- 
tions the same in height, or do they increase in height for sev- 
eral beats, forming the "stair-case contractions"? This can 
only be obtained after the ventricle has been inactive for a 
few minutes. Observe that the improvement in the degree of 
contraction in the "staircase contractions" occurs without any 
increase in the strength of the stimuli. 
87. Effect of a Weak and of a Strong Stimulus: When no 
further increase in the height of the contractions is obtained 
by repeating the minimal break shock, increase the strength of 
the stimuli by gradually shoving the secondary nearer to the 
primary coil. Observe that all the contractions are maximal 
and that a gradual increase in the intensity of the electrical 
stimulation does not cause any greater degree of contraction 
than a minimal stimulus. Unlike voluntary muscle, heart 
muscle does not show submaximal contractions from weak 
electrical stimuli. 
88. Latent Period and Time of Contraction: See that the 138 
Laboratory Exercises in Physiology. 
electro-magnet writes exactly on the same vertical line as the 
heart-lever when the drum moves at fastest speed. Set the 
drum for fastest speed and let it make one complete turn to 
get its maximum velocity. Stimulate with single induction 
shocks. Observe the long interval between the point of stim- 
ulation and the beginning of contraction, i. e., the "latent 
period." To measure this, connect the electro-magnet with 
the spring chronograph registering tenths of a second, and 
with the drum at the same speed as before, take a time tracing 
just below the other tracing. How long is the latent period in 
heart muscle? How much time is occupied by the periods of 
contraction and relaxation, respectively? Compare the time 
for a single contraction of heart muscle with that of voluntary 
muscle, giving the time occupied by the latent period, period 
of contraction and period of relaxation of each. 
89. Refractory Period: Arrange the electro-magnet to 
mark the point of stimulation exactly under the heart-lever as 
before. Find a strength of current which causes contraction, 
both at make and at break. Set the drum for fastest speed 
and let it make one turn to get a uniform velocity. With the 
key in the primary circuit make and break the current (1) 
so that break shocks fall in the latent period; (2) take another 
tracing with break shocks following the make at longer inter- 
vals, but within the period of contraction; (3) take a third 
tracing with still longer intervals, i. e., let the break shock fall 
immediately after the crest of the contraction wave caused by 
the make shock, or just at the beginning of diastole. Observe 
that only those stimuli which fall during the relaxation are 
felt; those falling in the latent period and period of contraction 
have no effect, i. e., they fall in the "refractory period" of 
the heart when it is not sensitive to stimulation. How long 
is the refractory period? 
Voluntary muscle is only refractory during the latent period; 
stimuli falling during the period of contraction cause a sum- 
mation or summing-up of the separate contractions. Can a 
summation of the contractions be obtained with the heart 
muscle ? 
90. Tetanizing Current: Find the minimal stimulus which 140 
Laboratory Exercises in Physiology. 
causes contraction, both at make and break. Arrange the in- 
ductorium for the tetanizing current. Shunt the electro-mag- 
net from the binding posts of the primary current. Set the 
drum at moderate speed and stimulate for about five seconds 
at a time. Does the tetanizing current produce a continuous 
contraction, like the complete tetanus in voluntary muscle, 
or a series of separate contractions? Explain the difference. 
91. Effect of the Galvanic Current: Remove the heart- 
lever, turn the heart up, and with scissors, sever the ventricle 
from the rest of the heart by cutting through the auriculo- 
ventricular groove. Place the excised ventricle on the cork 
plate and observe that it does not beat spontaneously. 
Disconnect the inductorium, but leave the simple key in the 
circuit from the two dry cells. Wrap the free end of each 
wire about a pin. Stick the pin connected with the positive 
pole of the battery in the cork plate so that it touches the 
base of the ventricle; stick the other pin in the cork so that 
it touches the apex, taking care not to prick the heart with 
the pins. 
Now close the key for a few seconds at a time and observe 
that when the galvanic current travels from base to apex, the 
ventricle usually gives a series of contractions during the pas- 
sage of the current. 
Reverse the direction of the current by changing the wires 
at the binding posts of the batteries. Again stimulate for a 
few seconds at a time. Usually when the current passes from 
apex to base, contractions only occur at the make and break 
and not during the passage of the current. If satisfactory re- 
sults are not obtained repeat the experiment with a weaker 
current. 
(Voluntary muscle only contracts at the make and break 
of weak or moderate galvanic currents.) 
NERVOUS CONTROL OF THE FROG'S HEART. 
92. Inhibition by Stimulation of the Crescent: Pith a frog 
and pin it out on the plate. Tie a ligature to the apex of the 
heart and connect with the short arm of a Gaskell lever to 
record the beats as in the previous experiments. 142 
Laboratory Exercises in Physiology. 
Arrange the inductorium for a tetanizing current with the 
electro-magnet shunted off from the primary binding posts and 
the hand electrodes attached to the secondary coil. Arrange 
the electrodes in the holder so that the platinum tips are in 
contact with the crescent. Starting with a weak current grad- 
ually increase its strength by shoving up the secondary coil 
until a strength of current is found which causes inhibition of 
the ventricle when the hand electrodes are applied to the 
crescent at the junction of the sinus venosus and right auricle. 
Use the minimum current which will produce this effect and 
be careful not to use too strong a current. 
The crescent contains motor as well as inhibitory nerve cells 
and often inhibition can not be obtained by stimulation at the 
first application of the electrodes. Tn that case the position 
of the electrodes should be changed until a location is found 
which gives inhibition when stimulated with a current of 
suitable strength. When that location has been found, care 
should be taken not to change the position of the electrodes. 
With the electro-magnet writing directly under the heart- 
lever, set the drum at moderately slow speed and take a tracing 
of the normal heart beat. Then stimulate the crescent for four 
or five seconds using the same strength of current as before 
and keeping the electrodes in the same position, so as to obtain 
a graphic record of cardiac inhibition. If inhibition does not 
occur increase the strength of current slightly, but be ex- 
tremely careful that the hand electrodes are applied to the 
crescent and not to the wall of the sinus or the auricle. 
Let the heart rest a few minutes. Now stimulate again. 
(a) Does inhibition appear as soon as the stimulus is ap- 
plied or is there a "latent period"? 
(b) Does inhibition disappear the instant the stimulation 
ceases or is there an "after-effect"? (The after-effect may 
appear as partial inhibition instead of complete inhibition.) 
(c) Is the ventricle inhibited in systole or diastole? 
(d) Now apply 0.2 per cent atropine sulph. sol. to the 
heart by allowing it to fall drop by drop on the ventricle 
from a small pipette. After waiting a minute or two, take 
another tracing. What effect has the atropine produced on 
the rate of the heart beat? 144 
Laboratory Exercises in Physiology. 
Now stimulate the crescent with the same current used be- 
fore and with the electrodes in the same position. Do you get 
inhibition? Explain the result. 
Now connect the electro-magnet with the binding posts of 
the time-marking current. Set the drum at the same speed as 
before and take a tracing of seconds under the tracings made 
above. 
93. The Ligatures of Stannius Recorded by the Graphic 
Method: With the heart arranged for recording the beats by 
the Gaskell lever, as in the last experiment, pass a silk thread 
beneath the two aortoe and bring it in position over the 
crescent. Take a normal tracing with the drum at moderately 
slow speed. Now tie the first ligature of Stannius, i. e., over 
the crescent, just at the junction of the sinus venosus and au- 
ricle. Observe that the sinus continues to beat as before, 
while the auricles and ventricle stop. 
Bring the lever to write very lightly on the drum and see 
that it is carefully counterpoised. Take a tracing of the sinus 
beats which shows slight waves in the same rhythm as the 
normal heart beats. 
Now tie the second ligature of Stannius about the auricles, 
just a line or two to the auricular side of the auriculo-ventrie- 
ular groove. After waiting a short time, the ventricle starts 
to beat, either at long and irregular intervals, or very slowly 
in a rhythm of its own, and quite different from that of the 
sinus. Take a tracing to show the sinus and ventricular beats 
after the second ligature. The effect of the second ligature 
is variable and not constant like that of the first ligature of 
Stannius. After waiting a reasonable time, another ligature 
may be applied close to the last one if the ventricle does not 
start to beat. Care should be taken that the second ligature 
is not tied in the aurieulo-ventricular groove or the ventricle 
will not beat again spontaneously. 
With another frog arranged for recording the beats with 
the Gaskell lever as before, take a normal tracing and then 
apply the Gaskell clamp about the middle of the auricles. 
Observe that the sinus and auricles continue to contract in the 
normal rhythm, while the ventricle contracts in an independent 146 
Laboratory Exercises in Physiology. 
rhythm. Several beats of the auricles occur for each beat 
of the ventricle. Take a tracing of these beats. Connect the 
electro-magnet to the time-marking current and take a trac- 
ing of seconds beneath each of the above tracings. Use this 
same frog for the next experiment. 
\ 
94. Influence of Temperature on the Sinus-auricular and on 
the Ventricular Beats: Disconnect the ligature from the short 
arm of the Gaskell lever and pass it through the short piece 
of rubber tubing of large lumen for immersing the heart in 
fluid. Attach the ligature from the apex to the short arm of 
the Gaskell lever again and arrange it to write lightly on the 
drum so that the sinus auricular contractions are registered 
distinctly. 
Cool some Ringer's sol. in a beaker by placing it in a 
mixture of ice and salt. When the temperature has been re- 
duced to 10° C. fill the rubber cup from a pipette and keep 
the heart immersed in the Ringer's sol. for two or three min- 
utes, or longer if necessary, until the rate of the auricular con- 
tractions is distinctly reduced. Now take a tracing with the 
drum at the same speed as before, keeping the heart immersed 
in cold Ringer's sol. from the pipette while the tracing is taken, 
taken. 
Take another tracing in the same way after applying Rin- 
ger's sol. at 5° C. with the drum at the same speed. 
Warm some Ringer's sol. and apply it to the heart at 30° to 32° 
C. for several minutes as before, until there is a distinct increase 
above the normal rate of contraction. Take a tracing with the 
drum at the same speed as before, allowing the warm Ringer's 
sol. to flow from the pipette into the rubber cup wThile the 
tracing is taken. 
Observe the influence of heat and cold, respectively, on the 
sinus-auricular and ventricular beats, after independent 
rhythms have been produced by the Gaskell clamp. If all 
parts of the heart come to beat at the same rate in one 
rhythm after warm Ringer's sol. has been applied, the Gaskell 
clamp may be applied again. 
95. Cardiac Inhibition from Stimulation of the Vagus in the 
Tortoise: Fasten a pithed tortoise on a holder. Cut close to the 148 
Laboratory Exercises in Physiology. 
plastron in removing it. Dissect out the vagus in the neck, 
and place small electrodes under the right vagus. 
Open the pericardium and tie a ligature to the apex of the 
ventricle. Attach this to a Gaskell lever with a light spring 
or a piece of rubber arranged above the lever to make slight 
tension upon the heart. Keep the lever horizontal between 
contractions. 
Arrange the induction coil for a tetanizing current with the 
electro-magnet as a shunt in the primary current. Find the 
minimal strength of current which will cause complete inhi- 
bition of the heart. Set the drum at moderately slow speed. 
After taking a tracing of the normal beats, stimulate the vagus 
with currents of suitable strength to get (a) partial and (b) 
complete inhibition, noting the position of the secondary coil in 
each instance. 
Observe carefully the effect upon the heart beats: 
(a) In what phase of the heart cycle does inhibition occur? 
(b) Does inhibition occur as soon as the stimulus is applied 
or is there a "latent period"? 
(c) Does the inhibition (either partial or complete) con- 
tinue as an "after-effect" for some time after the stimulation 
ceased? 
(d) What is the character of the first contractions that 
occur after complete inhibition? 
(e) After the heart returns to its normal rate, are the indi- 
vidual contractions any higher than the normal ones, i. e., have 
the contractions been improved by vagus stimulation? 
Connect the electro-magnet with the binding posts of the 
time-marking current, and, with the drum at the same speed 
as before, record seconds beneath each of the above tracings. 
96. Effect of Nicotine on the Heart: With everything ar- 
ranged as in the last experiment, set the drum at the same 
speed and make the following observations: 
1. Apply one or two drops of nicotine chloride directly to 
the surface of the turtle's ventricle with the drum at the 
same speed as before. If an excess of nicotine is avoided, 
there is partial inhibition from the stimulation of the inhibitory 
fibers within the heart. Stimulate the vagus again, using the 
minimal current which will cause complete inhibition. 150 
Laboratory Exercises in Physiology. 
2. Apply several drops of nicotine until the rate is in- 
creased. Now stimulate the vagus again, using the strength 
of current required to produce complete inhibition before. 
This fails to produce inhibition, as nicotine paralyzes the ex- 
trinsic nerve fibers to the heart. 
Before removing the paper from the drum, take a time- 
tracing, marking seconds beneath each of the above tracings 
with the drum at the same speed as before. 
This experiment, in connection with the next one, shows that 
nicotine probably paralyzes the inhibitory fibers or their ter- 
minations, but does not paralyze the inhibitory cells. The 
cells probably act as relay stations and are connected with 
the extrinsic inhibitory fibers by arborizations with the nerve 
fibers of the vagus. 
97. Action of Pilocarpine and Atropine: 1. Stop the drum 
for a few minutes and apply pilocarpine muriate until the de- 
crease in rate shows partial inhibition. Take a tracing of this 
with the drum at the same speed as before. 
Stimulate the heart by mechanical irritation (by touching 
the ventricle with forceps or pithing pin). Observe that the 
heart is irritable during the inhibition produced by pilocarpine. 
2. Apply atropine until the inhibition is overcome. Let 
the drum continue at the same speed as before. Stimulate 
the vagus again, starting with the same strength of current 
used before and gradually increase it. Observe that inhibition 
cannot be produced. 
Pilocarpine stimulates the inhibitory ganglia within the heart. 
Atropine paralyzes these, as well as the inhibitory fibers, so 
that stimulation of the crescent in the frog after the applica- 
tion of atropine fails to produce inhibition. (See Exp., 92, d, 
page 142.) This can not be done in the turtle, as the inhib- 
itory nerve cells are not grouped together at one point. These 
ganglion cells are paralyzed in the turtle as in the frog, and 
cannot be stimulated by pilocarpine after atropine has acted 
on them. 
Before removing the paper from the drum take time trac- 
ings, marking seconds under each tracing with the drum at 
the same speed. 152 
Laboratory Exercises in Physiology. 
98. Reflex Removal of Cardio-inhibitory Tone by Degluti- 
tion: Count your partner's pulse for four (4) periods of fifteen 
seconds each and record the observations as the normal. 
While he slowly sips cold water count his pulse again for four 
periods of fifteen seconds each. Record your result and compare 
with the normal rate. 
Observe that the removal of cardiac inhibition produced re- 
flexly by swallowing causes a considerable, but momentary, in- 
crease in the rate of the heart's action. 
99. Reflex Cardiac Inhibition from Stimulation of the 
Abdominal Sympathetic: The brain of a frog should be de- 
stroyed by pithing but the medulla should not be injured. Lay 
the frog on its back and count the number of heart beats in one- 
half minute by watching the heart through the thin chest wall. 
Make a small opening with scissors through the abdominal 
wall to one side of the epigastric vein. With forceps draw out 
several loops of the intestine and with the handle of a seeker 
strike it repeated sharp taps while your partner counts the rate 
of the heart beats as before. Record the rate before and during 
the violent mechanical stimulation. 
It has been found that the sympathetic fibers from the abdomi- 
nal viscera convey these afferent impulses to the cardio-inhibi- 
tory center in the medulla. 
NERVOUS CONTROL OF THE MAMMALIAN HEART. 
100. Cardiac Functions of the Pneumogastric Nerves: For 
this experiment a dog will be used. The animal is anesthetized, 
and the carotid arteries and pneumogastric nerves exposed by a 
median incision in the neck. A cannula is placed in each carotid 
artery. One is connected with a mercury manometer, and the 
other with a membrane manometer. The pens of the two mano- 
meters are arranged to write on the same perpendicular line on the 
large kymograph. The electro-magnet, marking seconds, is ad- 
justed to write on the base line or abscissa of the tracing by the 
mercury manometer. The mercury manometer records the gen- 
eral changes in blood pressure and heart rate, but the inertia of 
the mercury prevents it from showing slight and rapid changes 154 
Laboratory Exercises in Physiology. 
in the force of individual heart beats. The membrant mano- 
meter is a more accurate means of recording changes in the 
force and character of the individual heart beats. The car- 
diac functions of the nerves are shown by stimulating them with 
a tetanizing current and by cutting them. 
1. Observe the cardiac inhibition and fall in blood pressure 
that results from stimulation of these nerves. Describe the effect 
of stimulating both nerves (a) with a weak and (b) with a strong 
current. Note the increase in the size of each wave on the trac- 
ing during the inhibition. This is due to the fact that more 
blood is forced out with each beat (the ventricle being overfilled 
during the prolonged diastole) but the force is diminished as is 
shown by the fall in pressure. Note that the inhibition only lasts 
a short time after the stimulation is withdrawn, and that the 
beats rapidly return to the normal size and rate. (The "after 
effect" from stimulating inhibitory fibers is much less pro- 
nounced than that which follows stimulation of the sympathetic 
fibers.) 
2. Observe that when each nerve is stimulated separately with 
the same strength of current, one (usually the left) produces a 
greater degree of inhibition than the other. 
3. Observe that cutting one vagus, as a rule, produces but 
little change in the rate, the inhibitory control through the other 
vagus being sufficient to prevent marked increase in either rate 
or pressure. 
4. Note the reflex cardiac inhibition when the central end of 
one cut nerve is stimulated; and the direct inhibition from 
stimulation of the peripheral end. This shows that the vagi con- 
tain cardio-inhibitory fibers that run in both directions-from 
the heart to the center (afferent) and from the center to the 
heart (efferent). Observe that in reflex cardiac inhibition from 
stimulating the central end of the cut vagus, the fall in pressure 
does not occur, or is very slight, and sometimes there.is a rise in 
pressure. This is because the vasomoter center is stimulated re- 
flexly at the same time, and by constricting the blood-vessels, 
prevents the fall in pressure which would otherwise occur. 
5. Observe the marked rise in pressure and increase in rate 
that occurs after cutting both vagi. The cardio-inhibitory center 
exerts a constant restraining influence over the heart, the inhib- 156 
Laboratory Exercises in Physiology. 
itory tone, and when this restraint is removed the heart "runs 
away." The rise in pressure results from the increased fre- 
quency of the heart's action. Note that the rise in pressure does 
not last long, but disappears in a few minutes, because the blood- 
vessels become adjusted by dilating so as to offset the increased 
action of the heart. 
6. Observe that when the central end of the vagus is stimu- 
lated after both vagi have been cut, there is no slowing of the 
heart; this shows that the vagi contain all the efferent eardio-in- 
hibitory fibers. The rise in pressure in this case is due to stimu- 
lation of the vasomoter center. 
7. Observe the action of pilocarpine in causing cardiac in- 
hibition when injected intravenously after both vagi have been 
cut. This is due to direct action on the inhibitory nerve cells 
within the heart. 
Observe the antagonistic action of atropine in removing the 
cardiac inhibition by causing paralysis of the inhibitory ganglion 
cells within the heart. Note that stimulation of the peripheal 
ends of the cut vagi fails to produce inhibition after the admin- 
istration of atropine. 
Make a protocol of this experiment, and write out a brief sum- 
mary of the cardiac functions of the pneumogastrics from what 
you have seen. 
101. Functions of the Cardiac Sympathetic Fibers: In dogs 
the sympathetic fibers that go to the heart leave the spinal cord 
by the second, third and fourth thoracic nerves, and pass to the 
stellate or first thoracic ganglion that lies on the head of the 
second rib; from here the cardiac fibers reach the inferior cervi- 
cal ganglion in two bundles, that pass around the subclavian 
artery, forming the loop or annulus of Vieussens. 
In order to show the functions of these fibers, a dog is anesthe- 
tized; the carotid arteries are connected with the mercury and 
membrane manometers as in the last experiment; a cannula is 
tied in the trachea for artificial respiration, and the first rib on 
each side is resected so as to expose the subclavian vessels and 
stellate ganglion. The loop of Vieussens can then be placed on 
the electrodes. 
The sympathetic nerves contain both accelerator and augmen- 158 
Laboratory Exercises in Physiology. 
tor fibers for the heart. Their distribution on the two sides is 
variable; usually the augmentor fibers predominate on the left 
side and the accelerator on the right side. 
1. Observe the effect of stimulating the left annulus of Vieus- 
sens. Note the slight increase in rate and the great augmentation 
in force. The augmentation is shown by the increase in the 
height of the waves on the tracing of the membrane manometer, 
i. e., the increase in pulse pressure. The mean general blood 
pressure, as shown by the mercury manometer, may not be in- 
creased. 
2. Observe the effect of stimulating the right annulus of 
Vieussens. Note the marked increase in the rate of the heart, ac- 
companied by some rise in pressure, and by little or no aug- 
mentation. 
3. Observe the long latent period when the cardiac sympat- 
hetic fibers are stimulated. 
4. Observe that both augmentation and acceleration last for 
a considerable time after the stimulation is withdrawn, i. e., the 
"after-effect" is much more pronounced from stimulation of 
sympathetic than that from stimulation of inhibitory fibers. 
Record the protocol of this experiment and write a brief sum- 
mary of the functions of the cardiac sympathetic fibers from 
what you have seen. 
The Mammalian Heart in Action: At the end of the vagus 
experiment the chest of the dog will be opened and the heart 
exposed, the animal being kept alive by artificial respiration. 
102. Nature of the Contraction in Heart Muscle: The con- 
traction of the heart is a single twitch, and not a tetanus. This 
can be shown by the "physiological rheoscope, " a nerve-muscle 
preparation from a frog. If such a preparation is held so that 
the nerve rests on the heart muscle, the frog's muscle will con- 
tract with a single twitch for each beat of the heart, the nerve 
being stimulated by the "current of action" that is developed 
in the heart muscle with each beat. If the heart beat were a 
"tetanus" (a prolonged, continuous contraction caused by a 
number of stimuli) the frog's leg would show a prolonged 
contraction, instead of a twitch that occupies only 0.1 second. 160 
Laboratory Exercises in Physiology. 
103. Observe the Mammalian Heart in Action: (1) Note 
the sequence of events in the heart while it is slowed by pilo- 
carpine. (2) Observe the change in shape during contraction, 
especially the narrowing in the transverse diameter. Note that 
the whole heart becomes more pointed or cone-shaped in systole, 
and globular in diastole. The exposed heart is flattened out in 
diastole, but in the normal condition with the chest closed, this 
does not occur. (3) Observe the tilting up of the apex, the 
twisting of the heart from left to right and the descent of the 
auriculo-ventrieular groove during contraction. (4) Observe 
the hardening of the heart muscle. What relation has this phe- 
nomenon to the cardiac impulse? 
104. Time of Filling of the Coronary Arteries: Note that 
when a branch of a coronary artery is cut, the spurt occurs with 
the systole of the ventricle, just as in any other artery. 
105. Fibrillary Contraction: This is a condition in which 
the individual bundles of the heart muscle contract independ- 
ently of each other, so that coordination of contraction is lost and 
the heart looks like a quivering mass of jelly. Fibrillary con- 
traction can be produced by (1) tying one of the large coronary 
arteries or (2) by injecting milk (or melted paraffin) into one 
of the large arteries to produce embolism of the terminal coron- 
ary arteries or (3) by pucturing the so-called Kronecker's "co- 
ordination center"-a spot in the ventricular septum about the 
junction of the middle with the basilar third. 
Write out a brief account of the experiment to show fibrillary 
contraction. 
106. Influence of the Inorganic Salts of the Blood Upon the 
Heart: Pith a frog and expose the heart. Carefully cut the 
pericardium away from the two aorta1, taking care not to cut 
the two superior vena* cavte. Cut the frenum. Tie a silk ligature 
to the apex of the heart for attaching it to the Gaskell lever. 
Now turn the apex of the heart upward, making slight traction 
upon the silk ligature attached to it. Pick up the posterior layer 
of the pericardium with forceps and carefully dissect it away 
from the inferior vena cava so as to expose this vein below the 
point where it opens into the sinus venosus. By means of a 162 
Laboratory Exercises in Physiology. 
threaded blunt seeker pass a soft ligature about the inferior 
vena cava near the sinus venosus, pushing the seeker through 
liver tissue if necessary to get behind the vena cava. With the 
points of scissors make a nick in the inferior vena cava as far 
away from the sinus venosus as possible, and allow the blood to 
escape. Fill the T cannula and rubber tubing attached to it with 
Ringer's Sol. Close the end of the longer tube with a piece of 
glass rod, and apply a clip to the side tube; see that all air bub- 
bles are excluded. 
Now remove the blood and clots from about the heart by 
sponging with a piece of cotton moistened with Ringer's Sol. 
Insert the cannula into the opening in the inferior vena cava, 
and tie the soft ligature about its neck. After having tied the 
ligature about the neck of the cannula, tie the ends of the liga- 
ture around the side arm of the T cannula to keep it from pull- 
ing out. Turn the heart back to its normal position and with 
scissors make a nick in each aorta, close to the bulbous arteriosus. 
Fasten the frog plate to the stand and adjust the ligature 
attached to the apex to the short arm of the Gaskell lever; ar- 
range this to write lightly upon the drum; connect the electro- 
magnet to the time current to mark every fifth second. Connect 
the rubber tubing on the long arm of the cannula with the thistle 
funnel, taking care that there are no twists. The funnel should 
be on the level with the heart or a little above it, so that when it 
is filled the pressure will not overdistend 'the heart. The fluid 
in the funnel should be high enough to fill the ventricle and es- 
cape from the aorta without overdistending the auricles. 
1. Perfuse the heart with normal Ringers Sol. ^NaCl 0.7 
per cent, CaCl2 0.026 per cent, KOI 0.03 per cent.) Arrange the 
gear of the kymograph for slow speed and set the drum at such 
speed that the separate tracings of contractions can be counted. 
Take a short tracing of the normal (two or three inches). 
2. Replace the Ringer's Sol. with physiological salt solution, 
Sol. C (0.7 per cent NaCl). Do not stop the drum or change 
anything, but mark the point at which the change of fluid was 
made. Observe the steady decrease in the height of the con- 
tractions when the heart is perfused with an isotonic solution. 
3. As soon as the contractions cease, change the fluid to phy- 
siological salt solution, plus the normal amount of calcium found 164 
Laboratory Exercises in Physiology. 
in the blood, i. e., NaCI 0.7 per cent plus CaCl2 0.026 per cent 
(Sol. D). Mark the point at which the change was made but do 
not stop the drum or change the apparatus. 
4. As soon as the height of the contractions returns to nor- 
mal, observe whether or not the beats are rhythmical. Now 
change to physiological salt solution containing an increased per- 
centage of calcium, i. e., 0.06 per cent CaCl2 (Sol. E). Mark 
the drum to show the point at which the change of fluids was 
made as before. Observe the stimulating effect of the Ca upon 
the height of the contractions, especially the ventricular contrac- 
tions. If the tracing is continued long enough, the heart's action 
will become arhythmical from the excessive amount of Ca and on 
account of the absence of the K. Contractions occur after incom- 
plete relaxation of the heart muscle and "calcium spasm" may 
occur, i. e., more or less continuous contraction. * 
5. Restore the normal action of the heart again by perfusing 
with normal Ringer's Sol. (Sol. A). 
If the heart beats are restored to normal by perfusion with 
Ringer's Sol. (Sol. A.) the same frog may be used for the follow- 
ing experiments, or a fresh frog may be used arranged for per- 
fusion as before. In the either case take a tracing of the normal 
beats while perfusing with normal Ringer's Sol. (Sol. A.). 
6. Without stopping the drum or changing the position of 
any of the apparatus, remove the normal Ringer's Sol. by open- 
ing the clip on the side arm of the T cannula, and replace it with 
a modified Ringer's Sol. containing a high percentage of potas- 
sium, Sol B (NaCI 0.7 per cent, CaCl2 0.026, and KC1 0.07 per 
cent). Open the clamp on the side arm of the cannula and let 
some of the fluid escape; if necessary fill the funnel again with 
Sol. B. Mark the point on the drum at which the fluid was 
changed. ' 
Observe the potassium inhibition shown by the decrease in the 
rate and in the height of the contractions. As soon as this re- 
sult is obtained, replace the fluid in the funnel with normal 
Ringer's Sol. again; let a little escape by the side tube as before, 
and mark the position on the drum at which the change was made. 
Allow the perfusion with normal Ringer's Sol. (Sol. A.) to con- 
tinue long enough to remove the excess of potassium, i. e., until 
the heart beats become normal in rate and force. 166 
Laboratory Exercises in Physiology. 
7. Now replace the Ringer's Sol. in the funnel with a modi- 
fied Ringer's Sol. (Sol. F) containing the normal amount of 
calcium and potassium, but no sodium. This solution is made 
isotonic with 0.7 per cent NaCI by using a 2.15 per cent solution 
of dextrose or a 4.09 per cent solution of sucrose instead of sodi- 
um chloride. 
Observe that the beats of the heart gradually become weaker 
after the perfusion has been continued for a few minutes. Note 
the effect upon the muscle tone as indicated by the position of 
the heart lever at the end of the relaxation with each diastole. 
From these observations state your conclusions concerning 
the important part played by NaCI in the blood and in Ring- 
er's sol. Can it be replaced by a non-electrolyte in sufficient 
amount to give an isotonic solution, or does it serve some other 
purpose than that of maintaining a certain osmotic pressure? 
107. Influence of Drugs upon the Heart as Determined by 
Perfusion: When the heart beats have been restored to nor- 
mal by perfusing with normal Ringer's sol. after the preceding 
and each of the following experiments, make a tracing while 
perfusing with each of the following solutions: 
(a) A one-tenth saturated solution of ether in Ringer's 
sol., made by adding 9 parts of normal Ringer's sol. to one 
part of the same solution that has been fully saturated with 
ether. Care must be used to keep the bottle stoppered and 
to prevent the ether from vaporizing. 
(b) Perfuse with a one-tenth saturated solution of chloro- 
form in Ringer's sol. made in the same way as in the pre- 
ceding experiment. 
Observe that ether does not weaken the heart beats in this 
concentration, while chloroform causes marked direct depres- 
sion. 
(c) Perfuse with 1 to 50,000 sol. of adrenalin in Ringer's 
sol. and observe the stimulating effect upon the heart muscle. 
The action of digitalis and stophanthus may be shown in the 
same way. 
108. Isolation of the Mammalian Heart: For this experi- 
ment a dog or cat is used. The animal is anesthetized and the 
chest opened under artificial respiration; the heart is exposed 168 
Laboratory Exercises in Physiology. 
by opening the pericardium. The inferior and superior venae 
cavae are tied off, and a cannula placed in the aorta just above 
the semilunar valves. The heart is then cut out, and the 
cannula in the aorta attached to a tube leading from a bottle 
in which the perfusing fluid is kept under pressure. The per- 
fusing fluid consists of Ringer's solution which has been thor- 
oughly charged with oxygen. This is kept at bodily tempera- 
ture by immersing the pressure bottle in a water bath. Suffi- 
cient pressure is maintained in the bottle to force the fluid 
through the coronary circulation. This is done by allowing 
oxygen to escape from a cylinder into the large bottle con- 
taining Ringer's solution under a pressure equal to that of the 
blood in the coronary arteries, viz., 75-80 mm. Hg. This pres- 
sure closes the semilunar valves, and at the same time forces 
the fluid through the coronary vessels of the heart. The fluid 
after passing through the coronary vessels is returned to the 
right auricle, and is collected from a cannula tied in the 
pulmonary artery. The heart is immersed in Ringer's solu- 
tion, kept at bodily temperature. Observe the effect of changes 
in temperature of the Ringer's sol. Write out a brief sum- 
mary of the conditions necessary for the regular action of the 
isolated mammalian heart. IIow do the conditions differ from 
those necessary for the activity of the frog's heart? 170 
Laboratory Exercises in Physiology. 
THE BLOOD-VESSELS. 
PHYSICS OF THE CIRCULATION. 
109. Schema of the Circulation: This is an arrangement of 
glass tubes of different sizes to show (1) that division of the 
tubes increases the resistance and (2) that the velocity de- 
creases inversely in proportion as the combined sectional area 
of the tubes increases. 
Fluid flows from a pressure bottle through a large tube 
10 mm. in diameter; by means of Y's this divides into eight 
tubes of medium size, each 5 mm. in diameter; these in turn 
divide into sixteen small tubes, each of which has a diameter 
of 3 mm, and these divide into 36 tubes, each having a diameter 
of 2 mm. The tubes then unite by means of Y's at the other 
end so that the outflow from the system occurs through a single 
tube of the same size as the inflow. 
Vertical tubes are placed at different points to show the 
pressure (a) in the larger tubes representing the arteries; 
(b) in the smallest tubes which represent the capillaries; and 
(c) in the distal tubes which correspond to the veins. 
The area of transverse section of the single 
large tube (10 mm. diam.) = 78.5 sq. mm. 
Combined sectional area of 8 tubes of me- 
dium size (5 mm. diam.) =157.0 sq. mm. 
Combined sectional area of 16 small tubes 
(3 mm. diam.) =113.09 sq. mm. 
Combined sectional area of 36 small tubes 
(2 mm. diam.) =113.09 sq. mm. 
The frictional area, or area of contact between the fluid and 
wall of the tube, for a length of 1 cm. is as follows: 
Single large tube (10 mm. diam.) = 314.0 sq. mm. 
Eight tubes of medium size (5 mm. diam.). .=1256.6 sq. mm. 
Sixteen tubes of smaller size (3 mm. diam.). .=1523.9 sq. mm. 
In 36 tubes of smaller size (2 mm. diam.). .=2261.95 sq. mm. 
Observe (1) that air bubbles or suspended particles in the 
fluid move slowly in the small tubes, where the combined sec- 172 
Laboratory Exercises in Physiology. 
tional area is large as compared with the large tube where the 
sectional area is much less; (2) the pressure steadily diminishes 
as the distance from the pressure bottle increases; (3) the 
greatest fall of pressure occurs with the large number of small 
tubes, as division of the tubes causes a great increase in the 
area of contact between the fluid and the wall or the "fric- 
tional area." Make a protocol of this experiment. 
110. Flow Through Capillary Tubes: Record the time re- 
quired for a given amount of water to flow through glass tubes 
of the same length, but varying in diameter. Observe that the 
velocity of outflow is in inverse proportion to the square of the 
diameter if the diameter is more than 1 mm. 
Contrast that with the flow in capillary tubes of 1 mm. 
or less in which the time of outflow is inversely in proportion 
to the fourth power of the diameter. Record the time for each 
and explain the result. 
111. The Viscosity of Blood: Record the time required for 
(a) distilled water and (b) for defibrinated blood to flow 
through a viscosimeter. Exactly the same quantity of fluid 
is used at the same temperature in each case. How does the 
viscosity of blood influence its flow through a tube of capillary 
bore as compared with a limpid fluid like water? 
112. Harvard Circulation Schema: This is a mechanical 
device in which the fluid is propelled by a pump with valves 
which works intermittently to represent the heart; a piece 
of bamboo, with very small channels, offers an enormous peri- 
pheral resistance; manometers are connected with the tubes 
which correspond with the arteries and veins respectively; 
elastic tubes cause a continuous flow from the intermittent 
pressure. A side tube with a screw clamp enables one to vary 
the peripheral resistance, while a changeable eccentric on 
the pump allows variations to be made in the force with which 
the fluid is expelled. Changes in the arterial pressure from 
increasing or decreasing the force or frequency of the beats 
are easily produced; also the changes in the arterial and 
venous pressure from increasing or decreasing the peripheral 
resistance; pronounced pulsations may be felt by applying the 174 
Laboratory Exercises in Physiology. 
fingers to the rubber tube near the pump, but the flow from 
the veins is free from pulsation if considerable resistance is 
interposed. 
113. Flow of Fluids Through Rigid and Elastic Tubes: A 
rigid glass tube and an elastic rubber tube are connected with 
the two arms of a Y tube. The tubes have the same lumen 
and at their ends are attached tips of the same size. Observe 
(1) that the outflow from each tube is the same when the 
fluid is forced through them under constant pressure, as from 
a pressure bottle; (2) that when fluid is forced through the 
rigid glass tube intermittently, as from the pressure of a rub- 
ber bulb, the outflow is also intermittent, corresponding to the 
inflow; when fluid is forced into the elastic tube intermittently, 
the outflow is constant with spurts from each increase of 
pressure; (3) that the outflow from the rigid tube with inter- 
mittent pressure becomes continuous when an inverted bottle 
filled with air is connected to a T tube between the bulb and 
glass tube. The elastic recoil of the compressed air causes 
the flow between the spurts. 
Explain the cause (a) of the increased outflow from the elas- 
tic tube with each spurt and (b) of the flow between the 
spurts. What is the advantage of elastic over rigid tubes 
when the fluid is forced into them intermittently? 
114. Velocity of the Pulse Wave: Two levers with their 
pens writing on the drum in the same perpendicular line are 
arranged so as to rest at different points on a long piece of 
elastic rubber tubing; the length of tubing between the levers 
is three meters. Pressure from a rubber bulb causes an elastic 
wave to pass over the tube, while the drum is moving at fastest 
speed. With the drum moving at the same speed, a time trac- 
ing is taken in hundredths of a second by a tunipg fork. 
Observe (1) that the distal lever rises very soon after the 
proximal one. Note the interval of time between the two 
tracings, i. e., the time required for the wave to travel three 
meters, and determines the velocity per second. (2) Observe 
that the height of the distal wave is much less than the 
proximal one. (3) Contrast the velocity of the pulse wave 
with that of the current. Fluids of different colors are in turn 176 
Laboratory Exercises in Physiology. 
pumped through. Note the time that elapses from the time 
the fluid enters until it appears at the outflow. (4) That the 
pulse wave is independent of the flow of fluid is further shown 
by closing off the outflow. The pulse wave still travels over 
the entire length of the tube with each increase of pressure 
from the bull). 
METHODS OF RECORDING ARTERIAL BLOOD-PRES- 
SURE IN ANIMALS. 
115. (a) Hales' Method: This consists in connecting an 
artery of the animal (e. g.. carotid artery of a dog) with a 
long piece of glass tubing that is held in a vertical position. 
The tube is filled with Ringer's sol. The pressure is measured 
by the height to which the fluid is forced in the tube when the 
connection between the artery and tube is completed by re- 
moving the clip from the artery. The result is expressed in 
centimeters of Ringer's sol.; and this may be converted into 
terms of an equivalent column of water by multiplying the 
height of the column of Ringer's sol. by its specific gravity; 
this, in turn, may be converted into terms of a column of 
blood or of mercury by dividing by their respective specific 
gravities. 
Observe the great height to which the fluid is forced in the 
tube and the marked fluctuations that occur with the respi- 
ratory movements and with each heart beat. Record the re- 
sult of the experiment and convert it into mm. of mercury. 
This means of measuring blood-pressure is mainly of historical 
interest, it being the first method used. It also impresses one 
with the great amount of force required to displace the heavy 
mercury in a mercury manometer. 
116. (b) By the Mercury Manometer: This consists of 
a U-shaped piece of glass tubing containing mercury. One 
limb of the U-shaped tube is connected with the artery by heavy 
walled tubing (filled with some fluid that delays coagulation 
of the blood as a 2 per cent solution of sodium citrate). In 
the distal limb of the manometer a float rests on the surface of 
th^ mercury carrying a pen that writes on the drum. The 178 
Laboratory Exercises in Physiology. 
abscissa or base line is the line made by the pen before the 
manometer is connected with the artery. The electro-magnet, 
marking seconds, is arranged so as to write on this line. When 
the artery is connected with the manometer, the pressure of 
the blood forces the mercury down in the proximal limb, 
and up in the distal limb of the manometer, so that the pres- 
sure is measured by taking twice the height of the tracing 
above the abscissa. The mercury manometer is used for re- 
cording and measuring mean arterial pressure. This is cal- 
culated from the tracing by multiplying the distance from a 
point midway between the top and bottom of the curves to the 
abscissa by twro. The result is expressed in millimeters of 
mercury. 
117. (c) By the Huerthle Membrane Manometer: This 
consists of a small shallow tambour (about 15 mm. in diameter) 
covered with an elastic membrane. A small disc carrying a 
recording lever and pen rests on the membrane. A rigid style 
marks the base line. The chamber of the tambour is connected 
by heavy tubing with an artery, the tubing being filled with 
a 2 per cent sol. of sodium citrate to prevent clotting of the 
blood. The elasticity of the membrane has been calibrated 
against a mercury manometer so that the height of the pres- 
sure tracing may be expressed in millimeters of mercury. The 
mercury manometer is better suited for recording slight changes 
in the mean pressure, but the inertia of the mercury prevents 
an accurate registration of the rapid change in pressure with 
each heart beat. The membrane manometer follows more ac- 
curately the sudden variations in the pressure that take place 
with each heart beat. For this reason, the membrane mano- 
meter is used to determine the systolic pressure, i. e., the 
maximal arterial pressure during systole, and the pulse pres- 
sure, i. e., the increase in pressure which takes place between 
diastole and systole. The diastolic pressure is that which is 
maintained in the over-distended arteries between the systoles, 
i. e., during diastole. The pulse pressure is determined by 
subtracting the diastolic from the systolic pressure. 
Observe the height of the contractions recorded by the 
membrane manometer as compared with the small waves on 
the tracing taken by the mercury manometer. 180 
Laboratory Exercises in Physiology. 
In the experiment which yon have seen, record the mean 
arterial pressure, as determined by the mercury manometer, 
and find the equivalent of it in a column of water. 
Record the systolic, diastolic and pulse pressures, respect- 
ively, stating what each one is from what you have seen. 
118. Venous Pressure: The pressure in the veins is not 
sufficient to displace a heavy column of mercury, so that a 
water manometer* is used. The manometer and tubing con- 
necting it with the vein are filled with a dilute solution of 
sodium citrate. A T-shaped cannula is placed in a large vein, 
like the external jugular of a dog, where two branches of 
the vein join, so that the lateral pressure may be measured as 
the blood passes through the vein. (1) Observe and record 
the mean venous pressure in mm. of sodium citrate sol. when 
the clips are removed from the vein on both sides of the can- 
nula. Convert this into mm. of mercury. (2) Observe and 
record the changes in venous pressure (a) with inspiration 
and (b) with expiration. What causes the variations in venous 
pressure with the different phases of respiration? (3) Ob- 
serve and record the pressure in the distal end of the vein 
when the proximal end is occluded by a clip. (4) Observe and 
record the pressure in the proximal end when the distal end 
is occluded. 
119. Influence of Saline Infusion on Arterial and Venous 
Pressure: Connect the cannula in the vein with a mercury 
manometer which writes immediately under the mercury 
manometer recording the arterial pressure. The tubing be- 
tween the vein and manometer is filled with sodium citrate 
solution. 
After taking a tracing of the normal, Ringer's sol, is in- 
jected intravenously at the bodily temperature in such quan- 
tity that the volume of blood is increased 50 per cent. Observe 
the increase of venous pressure without any increase of ar- 
terial pressure; also the rapid return of the venous pressure 
to normal. 
After the return to the normal, the volume may be increased 
100 or 200 per cent without increasing the arterial pressure. 
Explain the results. 182 
Laboratory Exercises in Physiology. 
120. Influence of Posture on Blood-Pressure: The blood- 
pressure is taken in the carotid artery. After the animal has 
recovered from the anesthetic and regained consciousness, what 
is the effect upon blood-pressure (in the carotid) of (a) raising 
the feet? (b) Of raising the head and lowering the feet? 
The animal is then etherized again and kept in a condition 
of surgical anesthesia, (a) What is the effect upon blood- 
pressure of raising the feet in the state of deep anesthesia?1 
(b) What is the effect of raising the head and lowering the 
feet? Explain the difference between the two conditions and 
state why the posture during anesthesia is important. 
121. Effect of Breeding and of Transfusion upon Blood- 
Pressure: What is the effect upon the arterial pressure of 
removing from one-fourth to one-third of the animal's blood 
by bleeding from the distal end of the jugular vein? 
Observe the change in the pulse pressure as the mean arterial 
pressure falls. Does the mean pressure improve before trans- 
fusion is started? Explain how this is brought about. 
The blood is rendered non-coagulable by mixing with sodium 
citrate in the proportion of 0.25 per cent and injected at the 
bodily temperature. Observe that the restoration of the vol- 
ume of blood by injecting the citrated blood causes the arterial 
pressure to return to normal. 
The animal is then bled again to the same extent as the 
first time and mixed with sodium citrate sol. as before. 
Now inject an equivalent amount of. Ringer's sol. at the 
bodily temperature. Observe that the arterial pressure is 
not completely restored to the normal as Ringer's sol. does 
not have the same viscosity as blood. The subsequent injection 
of the citrated blood, or of Ringer's solution made viscid by the 
addition of a colloid will restore the pressure. 
Record the results and explain what becomes of the excess 
of fluid after saline infusion. 
122. Influence of Respiration upon the Pulmonary Blood- 
Vessels: 1. The lung of a frog is exposed through an incision 
in the flank and arranged so that the vessels may be observed 
directly with the low power of the microscope. The lung is 184 
Laboratory Exercises in Physiology. 
distended by positive pressure from within, by means of a 
cannula tied in the larynx. 
Observe that the blood-vessels are narrowed in direct pro- 
portion to the distending force of the air within the lung. 
Extreme distension causes complete blood stasis. 
Note that this condition of pulmonary vasoconstriction ob- 
tains during the inspiratory phase of artificial respiration in 
animals; also that a similar condition occurs in certain forms 
of emphysema of the lungs and explains the hypertrophy and 
dilatation of the right heart. 
2. In another frog the lung is placed in a glass bulb with 
a narrow opening. The lung is distended by negative pressure 
in the bulb outside the lung, similar to the negative intra- 
thoracic pressure in mammals. 
Observe the great dilatation of the blood-vessels when the 
lung is distended in this way. Note that this is the condition 
of the lung and pulmonary vessels during the inspiratory 
phase of natural respiration. This widening of the pulmonary 
blood-vessels allows more blood to get through from the right 
to the left heart during inspiration. 
123. The Influence of Respiration on the Arterial Pressure: 
The variations in arterial pressure with the different phases of 
respirations are just the reverse of the changes in the venous 
pressure. During most of the phase of inspiration the arterial 
pressure rises, due to a dilated condition of the pulmonary ves- 
sels which allows more blood to get through from the right to 
the left heart. During the greater part of expiration the arterial 
pressure falls as less blood can get through the constricted pul- 
monary vessels and return to the left heart. 
The respiratory movements are registered by a tambour con- 
nected with the trachea. The negative intratracheal pressure 
shows the time occupied by inspiration, while the positive pres- 
sure occurs with expiration. The tambour writes oh exactly the 
same vertical lines as the tracing of the arterial pressure by the 
mercury manometer. 
If the respiratory rate is rapid it may be difficult to determine 
the exact time of the changes in the blood pressure. When both 
vagi are cut, or when the trunk of the nerve is frozen so as to 186 
Laboratory Exercises in Physiology. 
block the nerve impulses and thus prevent them from reaching 
the respiratory center from the lungs, the respirations become 
very slow and the changes in arterial pressure caused by the 
different phases of respiration can be followed. It sometimes 
happens that there are no respiratory curves on the tracing of 
the arterial pressure. They usually become more prominent 
when the air encounters considerable resistance in entering and 
leaving the lungs. 
1. Observe that the natural respiratory movements and the 
large curves on the tracing of the arterial pressure do not exactly 
coincide. Inspiration begins just before the pressure reaches 
its minimum, and ends just before the maximum pressure oc- 
curs. Expiration follows at once and most of the fall in pres- 
sure occurs during this phase of respiration. 
2. In artificial respiration, as used with animals in the lab- 
oratory, the conditions are reversed. Air is forced into the lungs 
under a positive pressure in inspiration, causing a constriction 
of the pulmonary vessels and a consequent fall of arterial pres- 
sure. During expiration the air escapes with the return of the 
distended thorax to the normal and the blood pressure in the 
arteries increases as more blood gets through the lungs. Thus 
the variations of arterial pressure with artificial respiration are 
the opposite of those with natural respiration as the mechanism 
is totally different. 
3. Positive ventilation of the lungs or inflation with com- 
pressed air forced into the trachea, under sufficient pressure to 
produce moderate distension of the thorax, causes inhibition of 
the respiration and a fall of arterial blood pressure due to the con- 
striction of the pulmonary blood vessels. The extent to which 
the arterial pressure falls depends upon the amount of intra- 
pulmonary pressure exerted by the air in distending the lungs. 
The arterial pressure may be reduced to one-third or one-fourth 
of the normal by producing considerable distension of the lungs 
and thorax. The vascular change (constriction) seen in the 
frog's lung from inflation by means of a cannula in the trachea 
explains the tremendous fall of arterial pressure produced in the 
dog by the same procedure. 
4. Negative ventilation of the lungs may be produced by 
causing the animal to breathe rarified air. The tracheal cannula 188 
Laboratory Exercises in Physiology. 
is connected with a large vessel or tank containing a considerable 
volume of air under negative pressure of -15 to -30 mm. Hg. 
Observe that the breathing of air under less than the ordinary 
atmospheric pressure causes a rise of arterial pressure due to 
dilatation of the pulmonary vessels. Such change in arterial 
pressure does not occur in going from the sea-level with high 
atmospheric pressure to the low barometric pressure of a high 
altitude, because the peripheral vessels of the pulmonary and 
those of the systemic circulation are subject to the same change 
and one balances the other. 
It is clear that negative intrapulmonary air pressure causes 
the vessels of the lungs to dilate in the same way that the vessels 
of a frog's lung can be seen to be dilated when the lung is dis- 
tended in a glass chamber by a negative pressure outside the 
lung. 
(a) Explain the cause of the rise of arterial pressure during 
inspiration, and the fall with the expiratory phase of natural 
respiration. 
(b) Explain the respiratory changes of arterial pressure 
during artificial respiration as it is used in animals. 
(c) Describe the effect upon arterial pressure of breathing 
air from a pneumatic cabinet under positive and negative pres- 
sure, respectively, without exposure of the exterior of the body 
to the same atmospheric pressure. 
124. Influence of Asphyxia on Arterial Blood Pressure: 
When the air is prevented from entering the lungs so that the 
interchange of gases between the air and blood can not take 
place, the respiratory movements are at first very rapid (hy- 
pernea). The vigorous and struggling respiratory movements 
cause enormous respiratory curves on the tracing of the arterial 
pressure. Gradually the movements become less rapid but more 
labored (dyspnea) with active expiratory effort. This stage of 
excitement may end in convulsive movements. 
During this stage there is pronounced cardiac inhibition from 
stimulation of the cardio-inhibitory center. The blood pressure 
steadily rises, in spite of the cardiac inhibition, from direct 
stimulation of the vaso-constrictor center by the venosity of the 
blood. A general convulsion also causes rise of blood-pressure. 190 
Laboratory Exercises in Physiology. 
Sometimes the cardiac inhibition is sufficient to offset the vaso- 
constriction. When that is the case, the administration of atro- 
pine causes paralysis of the cardio-inhibitory cells and the rise 
of pressure from stimulation of the vaso-motor center can then 
be demonstrated. 
During the third stage, or that of collapse, the blood-pressure 
is low from paralysis of the vasomoter center; the respirations 
have ceased or only occur at long and irregular intervals; the 
heart continues for a considerable time after'respirations cease. 
The spontaneous respirations can be re-established and the blood- 
pressure can be restored to normal by lowering the head and us- 
ing artificial respiration. 
NERVOUS CONTROL OF THE BLOOD VESSELS. 
125 The Circulation in the Web of the Frog's Foot: Ob- 
serve (1) the velocity pulse in the arterioles; (2) the compara- 
tively slow current, without pulsations, in the small capillaries; 
(3) the venules are larger vessels with swifter current but free 
from pulsations. Observe that the corpuscles move much slower 
at the periphery than in the middle of the stream. From this 
fact, what would you consider the chief cause of the resistance 
encountered by the blood? 
Why is the velocity less in the capillaries than in the arteries 
and veins? 
Explain the cause of the continuous flow, without pulsations, 
in the capillaries. 
126. Efferent Vasomoter Fibers in the Sciatic: In a frog 
that has been lightly curarized the sciatic nerve is cut on one 
side. The webs of both feet are pinned out on a cork plate and 
placed on the stages of two microscopes placed side by side. In 
this way comparison can be made with the normal. (1) Observe 
the dilated blood-vessels and increased flow of blood which re- 
sults from section of the nerve. Explain the cause of this con- 
dition and tell how vasomotor tone is maintained normally. (2) 
Electrodes are placed under the peripheral end of the sciatic and 
the nerve is stimulated with a tetanizing current. Constriction 
in the individual vessels can not be seen, but the vascularity is 192 
Laboratory Exercises in Physiology. 
greatly decreased during stimulation and the flow is lessened or 
stopped in the capillaries. What change takes place in the ar- 
terioles during stimulation of the nerve? Does vasoconstriction 
occur in the arterioles or capillaries? Note that the vasodilata- 
tion soon returns after the stimulation ceases. 
127. Vasomotor Fibers of the Cervical Sympathetic: The 
ears of a rabbit are shaved and the cervical sympathetic is cut 
on one side. (1) What is the condition of the larger blood-vessels 
of the ear on that side? How does the pink color of the ear be- 
tween the blood-vessels compare with that of the other side? 
Explain the cause of this increased vascularity. (2) The peri- 
pheral end of the sympathetic is stimulated with a tetanizing 
current. What is the effect upon the blood-vessels of the ear of 
that side? (Comparison should be made with the normal side 
in each case.) 
128. The Depressor Nerve in the Rabbit: In the rabbit this 
is a branch of the vagus, found in the sheath of the carotid with 
the vagus and sympathetic, and consists of afferent vasodilator 
or depressor fibres. (1) Observe that stimulation of this nerve, 
with the vagi intact, causes a fall of pressure with pronounced 
cardiac inhibition. What conditions result from the stimulation 
of the nerve which may explain this fall of pressure? (2) 
The vagi are cut. Observe that stimulation of the nerve now 
causes just as great a fall of pressure without any cardiac inhibi- 
tion. Explain the cause of the lowered pressure in this case. 
(3) Observe that section of the nerve does not cause any change 
in the blood-pressure. (4) Observe that stimulation of the peri- 
pheral (cardiac) end has no effect, while stimulation of the cen- 
tral end causes as great a fall of pressure as was obtained when 
the unsevered nerve was stimulated. 
Is the vasodilatation which results from stimulation of the 
depressor nerve a local one, due to stimulation of efferent vaso- 
dilator fibers, or is it a general vasodilatation produced reflexly 
by stimulation of afferent vasodilator (depressor) fibers? Under 
what conditions would these nerve fibers be stimulated normally? 
Make a protocol of the experiment to show these points. 194 
Laboratory Exercises in Physiology. 
129. Vasomoter Functions of a Mixed Spinal Nerve, e. g., 
the Sciatic: (1) What is the effect upon general arterial 
blood-pressure (in the dog) of stimulating the sciatic nerve in- 
tact? Explain what conditions may cause the rise of pressure. 
(2) Observe that section of the nerve has no effect upon mean 
arterial pressure; neither does stimulation of the periphral end, 
although there is a local vasoconstriction, as was seen in the 
frog's foot. Explain why this local vasoconstriction fails to in- 
fluence the mean arterial pressure. (3) Observe that stimulation 
of the central end produces just as great a rise of pressure as was 
obtained at first. After section of the great splanchnic nerve 
there is little or no rise of pressure from stimulation of the cen- 
tral end of the sciatic. Explain how stimulation of the central 
end causes a rise of pressure. 
130. Vasomoter Fibers of the Splanchnic Nerve: The great 
(left) splanchnic nerve is exposed just above the diaphram by 
resecting a portion of the lower ribs in a dog. (1) What is the 
effect upon the blood-pressure and heart rate of stimulating the 
trunk of the nerve with both vagi intact ? Is the cardiac inhibi- 
tion sufficient to prevent a rise of pressure? (2) Observe that 
section of the great splanchnic in the dog does not cause a fall 
of pressure. (In the rabbit the pressure is lowered by cutting 
this nerve.) (3) Observe that stimulation of the central end of 
the cut splanchnic fails to cause a rise of pressure, while stimula- 
tion of the peripheral end causes as great an increase as before. 
Compare this with the stimulation of the sciatic nerve in the last 
experiment and explain the difference in the results obtained. 
(4) Both vagi are cut to sever the efferent cardio-inhibitory 
nerve fibers going to the heart. Observe that reflex cardiac inhi- 
bition can not be obtained by stimulating either end of the cut 
splanchnic after section of both vagi. (5) When the peripheral 
end of the splanchnic is stimulated with the same strength of 
current as before, observe that the rise of pressure is greater 
after section of the vagi than when they are intact, as there is no 
inhibition of the heart. 
Make a protocol of this experiment and write a brief sum- 
mary of the vasomotor function of the splanchnic nerve. 
131. Vasomoter Changes Produced by Drugs: (1) Adrenalin 196 
Laboratory Exercises in Physiology. 
is injected intravenously (10-20 c. c. of 1-20,000 Sol.). Observe 
the pronounced rise of pressure from the vasoconstriction which 
pases off in a few minutes. With the vagi intact, adrenalin 
often causes such a degree of cardiac inhibition that there is 
comparatively little increase in the mean pressure. Section of 
both vagi prevents the cardiac inhibition to a certain extent, but 
not entirely, as the drug appears to act upon the inhibitory nerve 
cells within the heart as well as the neurones in the medulla. 
If cardiac inhibition persists after section of the vagi, it may be 
removed by a full dose of atropine. 
Observe that the rise of pressure from adrenalin is very great 
when the vasoconstriction is not offset by cardiac inhibition. The 
pressure may amount to double or treble the average height. 
(2) The inhalation of amyl nitrite causes a pronounced fall 
of pressure due to vasodilatation. Observe that the effect soon 
passes off. 
(3) The influence of partial asphyxia upon the blood-pressure 
may be shown before and after cutting the spinal cord. The fact 
that rise of pressure from vasoconstriction only occurs while the 
spinal cord is intact shows that the vasomotor center is stimulated 
by the venosity of the blood. The best results are obtained when 
the first observation is made before the great splanchnic nerve 
is cut. 
The spinal cord is cut in the upper cervical region and artifi- 
cial respiration is maintained. Observe the great fall of blood- 
pressure which follows section of the spinal cord and explain the 
cause of it. 
Now adrenalin is given again in exactly the same dose as 
before. Observe that adrenalin produces relatively as great a 
rise of pressure as before. Of course, the pressure does not reach 
the same absolute elevation as in the normal condition. From 
this experiment what would you conclude as to the way vasocon- 
striction is produced by adrenalin ? 
132. Traube-Hering Curves: A dog that has had both 
vagi severed to remove the nervous control of the cardio-inhibi- 
tory centers upon the heart, has a tube tied in the trachea for 
carrying on artificial respiration. Curare is then injected into 
the vein slowly and cautiously until voluntary movements and 198 
Laboratory Exercises in Physiology. 
the respiratory movements cease. Care should be exercised to 
give just enough to paralyze the motor nerves, but no more, as 
an excess causes vasomotor paralysis. Artificial respiration is 
then established for a few minutes. The respiratory curves on 
the arterial pressure, if they now occur, show a fall during in- 
spiration and a rise during expiration, just the reverse of those 
seen in natural respiration. The tube through which the lungs 
are inflated is then detached from the tracheal tube for short 
periods of time while the drum is revolving. It will be seen 
that slow, rhythmical curves occur on the arterial pressure tracing 
although there is no respiratory or voluntary movements. These 
are known as Traube-Hering curves and are due to rhythmical 
activity of the dominating vasoconstrictor center. One of these 
occupies as much time as several respiratory curves. Sometimes 
curves occupies as much time as several respiratory curves. 
Sometimes they may be seen when respiration is going on. 
133. Circulation Time: This is determined (for class dem- 
onstration) by injecting 5 to 8 c. c. of a saturated solution of 
medicinal methylene blue into the veins at different points of 
the circulation, and, with a stop watch, noting the time that is 
required for it to appear at other points. Methyl blue is changed 
to a colorless substance in a few minutes in the blood so that the 
observations may be repeated a number of times. 
1. Observe the time that intervenes between the injection of 
methyl blue into the external jugular vein and its appearance 
in the carotid artery. , This gives the time required for a cir- 
«uit of the lesser circulation, and, in small dogs, it is not more 
than three or four seconds. 
•2. Observe the time required for the pigment to appear in 
the femoral artery after an injection into the jugular vein. 
3. Observe the time which intervenes between the injection 
of the pigment into the femoral vein and its appearance in the 
femoral artery. Note that this is very little more (about two 
seconds) than the time required for the lesser circulation. 
4. If the large branch of the femoral artery (profunda fe- 
moris) is large enough for the introduction of a cannula, an in- 
jection of methylene blue may be made into the femoral artery 
without interfering with the natural flow of blood through its 200 
Laboratory Exercises in Physiology. 
branches. Observe the time that intervenes between the injec- 
jection of methylene blue and the appearance of the blue color 
in the femoral artery of the opposite side. This represents the 
complete circulation time, i. e., the time of the lesser circulation 
plus the longest loop of the greater circulation. 
Record the results of two observations in each case. 
134. Measurement of the Velocity of the Blood Stream by 
Ludwig's Stromuhr: This is a U-shaped tube with bulbous 
enlargements of definite capacity; the ends of the tube are at- 
tached to the upper of two disks, so that the bulbs may be re- 
versed quickly without any leakage; two tubes from the lower 
disk enable the bulbs to be put in communication with both 
ends of a severed artery; these tubes are fitted with cannulas 
of different sizes, so that the instrument is connected with the 
artery by cannulas having the same lumen as the artery. 
The instrument measures the volume of blood that passes 
through a vessel of definite size in a given unit of time (one 
second). The velocity is determined by dividing this volume 
of blood by the area of the transverse section. 
A small dog is used and the coagulability of the blood de- 
stroyed by the intravenous injection of 0.001 gram, of Hirudin 
for every 8 c.c. of blood in the body. 
The proximal bulb (i. e., toward the heart) is filled with oil; 
the distal one with defibrinated blood. When the clips are re- 
moved from the artery the blood from the central end of the 
artery rises under the oil and displaces the defibrinated blood 
from the distal bulb into the peripheral end of the artery. 
When the oil has all been displaced to a certain mark on the 
distal bulb, the bulbs are quickly reversed and the one contain- 
ing oil is again placed in communication with the central end 
of the artery. 
Seconds are registerd on a revolving drum and an electro- 
magnet connected with the disk of the stromuhr, writes on the 
same vertical line as the second marker. Each time the bulbs 
are reversed, electrical contact is made by the disk and marked 
on the drum by the electro-magnet. The bulbs are reversed, 
as often as the oil is displaced, for one or two minutes, and an 
average taken. 202 
Laboratory Exercises in Physiology 
Having determined the average amount of blood that es- 
capes in one second, and the transverse section of the artery, 
the velocity is found by dividing the former by the latter. 
For instance, if 1 c.c., or 1000 c. mm., escapes per second, and 
the sectional area of the artery is 3.14 sq. mm., then the velocity 
is 318 mm. per second. 
135. Velocity in the Capillaries: This may be determined 
by direct observation of the flow in the web of the frog's 
foot, using a micrometer eyepiece and an electro-magnet to reg- 
ister the time of each observation on a drum moving at fast 
speed. A time-marker should register tenths of a second just 
beneath the electro-magnet. 
The eyepiece is arranged so that the micrometer scale coin- 
cides with the stream in as long a stretch of a capillary as 
possible. The vision is fixed on some one corpuscle, and with 
the hand on the key the time required for the corpuscle to 
travel from one part of the scale to another is indicated by the 
electro-magnet. The amplification being known, it is an easy 
matter to calculate the distance traveled per second. 
The average of a number of observations should be taken. 
In this experiment much depends upon the training and care 
of the observer. From a comparison of the velocity of the 
blood in the large arteries with the velocity in the capillaries, 
what would you conclude as to the combined sectional area 
of the capillaries compared with that of the arteries? 
136. The Volume Pulse: This may be determined (a) by 
using two small plethysmographs or glass tubes, into which two 
fingers are inserted with a rubber collar fitting tightly about 
them at the mouth of the tube. The rubber should not be 
tight enough to interfere with the venous circulation. 
The plethysmographs are then connected by means of a Y- 
tube and rubber tubing with a delicate recording tambour, 
with a T-tube interposed between the two. When everything 
is adjusted a spring clamp is placed on the side arm of the T. 
With each heart beat there is an increase in the volume of the 
finger and the pulsations are registered by the tambour. 
The drum must be smoked very lightly and the lever of the 
tambour must write with the least friction possible. By care- Laboratory Exercises in Physiology 
204 
fully counterposing the lever of the tambour, good tracings of 
the volume pulse can be obtained, showing a distinct dicrotic 
notch. 
(b) The large plethysmograph for enclosing the hand and 
forearm gives much larger pulsations than those obtained from 
the fingers. The hand is held in a special glass cylinder; the 
hand and forearm are surrounded by water at the temperature 
of the body; with each heart beat the water is displaced by the 
increase in the volume of the part, giving pulsations with the 
dicrotic notch in the usual position. These may be recorded 
by a tambour or by registering the changing level of the fluid 
as it is displaced from the plethysmograph. 
This instrument is used for determining the changes in the 
peripheral blood-vessels. With dilatation of the surface vessels, 
the volume of the hand increases greatly, while the opposite 
change occurs with vasoconstriction. 
(c) The oncometer: This is a special plethysmograph for 
measuring the volume of the kidney or spleen and the changes 
that take place with varying conditions of the blood-vessels. 
It consists of a metal box, made in two halves, each lined with 
delicate peritoneal membrane. The kidney is encased without 
interfering with its circulation or the outflow of urine; the 
space between the kidney and the box is completely filled with 
a light oil at the temperature of the body; this communicates 
with a piston recorder or an oncograph so that the increase in 
the volume of the kidney with each heart beat may be recorded 
on a revolving drum. The pulsations are very pronounced and 
show the dicrotic notch as distinctly as the tracings of the 
arterial pulse. 
Enormous changes in the volume of the kidney may be shown 
by producing general vasodilatation by the inhalation of amyl 
nitrite, and by producing vasoconstriction by the injection of 
adrenalin. Tn the former case the kidney expands and the 
oncograph tracing rises as the arterial pressure falls; in the 
latter, the volume decreases and the plethysmographic tracing 
falls as the arterial pressure rises. 
The same changes may be shown by cutting the splanchnic 
nerve of the same side to produce vasodilatation and by stimu- 206 
Laboratory Exercises in Physiology 
lating the peripheral end to cause vasoconstriction. The in- 
fusion of Ringer's Sol. also causes the kidney to swell by in- 
creasing the capillary pressure, although the arterial pressure 
is not changed. 
137. Arterial Pressure in the Frog: The frogs have been 
curarized and do not require pithing, except to destroy the brain 
without destroying the medulla. Pin the frog out on the frog 
plate; cut through the skin in the usual way from the pelvis to 
the jaw. Now tie the epigastric vein near the pelvis by making 
a nick through the abdominal wall on each side of it, and passing 
a ligature beneath it. Expose the heart in the usual way, and 
tie the frenum. Be careful to avoid hemorrhage. 
Clear the two aortas. Notice which is the larger (usually the 
left) ; pass a ligature beneath it and tie as far away from the 
heart as possible. Fill the cannula and tube with sat. sol. of 
sodium bicarbonate and close the end of the tube with a piece of 
glass rod. Place a small clip on the left aorta close to the bifurca- 
tion, and tie a second ligature loosely about the vessel between 
the clip and the first ligature. Make a small opening in the 
vessel between the first ligature and the clip, and insert the point 
of the cannula into it; let your partner fasten it in place by 
tying the second ligature about its neck. Fasten the frog plate 
to the stand at a little higher level than the manometer. See 
that the proximal limb of the jnanometer is filled with soda 
solution to the exclusion of all air bubbles. Arrange the electro- 
magnet so that its pen writes on the same horizontal line as the 
pen of the manometer before the rubber tube of the cannula is 
attached to it. Connect the electro-magnet with the time circuit 
to mark seconds. The line made by the electro-magnet will now 
mark the level of atmospheric pressure in the manometer, or 
the "abscissa," of the blood-pressure tracing. 
Now pinch the rubber tube behind the glass rod and remove 
it; fill the end of the tube with soda solution; attach it to the 
manometer before releasing the constricting fingers. Note that 
as soon as the tube is released the mercury in the proximal limb 
of the manometer falls, and rises correspondingly in the distal 
end, and that with each beat of the heart the mercury rises and 
falls. 208 
Laboratory Exercises in Physiology 
Take a normal tracing two or three inches in length with the 
drum at very slow speed. 
138. Effect of Cardiac Inhibition on Blood-Pressure: Ar- 
range the inductorium for the tetanizing current and attach the 
hand electrodes to the secondary. Find the strength of current 
that will cause marked inhibition when applied to the crescent. 
Set the drum going again at very slow speed; raise the heart 
gently and stimulate the crescent, your partner marking on the 
drum the beginning and end of the stimulation. Do not stop 
the drum until the pressure and rate have returned to normal. 
What effect has inhibition of the heart on blood-pressure? 
139. Effect of Stimulating a Sensory Nerve on Blood-Pres- 
sure: After waiting a few moments, set the drum going at 
the same speed as before, and with the hand electrodes, stim- 
ulate the brachial plexus (the single large nerve trunk going 
to the fore leg) on the side, opposite to that in which the 
cannula is tied in the aorta. Observe the rise in pressure. 
How is this produced? 
Effect of Destroying the Spinal Cord: Carefully turn the 
frog on its side and pith it; then destroy the cord by pushing 
the pin into the spinal canal. Return the frog to its original 
position and take a tracing. Observe that there is a marked 
fall in pressure. Why? 
After your tracings have been varnished and dried, measure 
the blood-pressure in each one. Mark the mean between the 
top and bottom of the curves and multiply the distance (in 
mm.) between this point and the abscissa by two. Record the 
results in mm. of Hg in a protocol of the experiment. 
140. Perfusion of the Blood-vessels: Pith a frog and de- 
stroy its brain by passing the pin anteriorly, but do not destroy 
the cord. Expose the heart in the usual way. Grasp the ven- 
tricle with forceps and with the points of scissors make a nick 
in the sinus venosus. Let the heart bleed freely for a few 
minutes, removing the blood and clots with moist cotton. 
Fill the T-cannula and tubing with Ringer's solution; close the 
short lateral tube with a clip, and the long tube with a piece 
of glass rod. Be sure to exclude all air bubbles from the cannula 
and tubing. 210 
Laboratory Exercises in Physiology 
Place a soft moist ligature about the auricles so that it can 
be tied over the bulbus arteriosus. Hold the ventricle with 
forceps and with the scissors make a small nick or V-shaped 
opening at the base of the ventricle or in the bulbous arteriosus 
close to its origin. Insert the cannula into the bulbus ar- 
teriosus and compress the rubber tubing gently to see that 
the bulbous arteriosus and aorta? are distended during the com-, 
pression. This makes sure that the cannula has not passed back 
into auricle through the tricuspid orifice. 
Let your partner fasten the cannula in position by tying the 
ligature about its neck and tie the ends of this ligature about the 
lateral arm of the cannula to keep it from pulling out. Hook a 
wire through the frog's nose, and hang it by the wire to the funnel 
ring. Remove the rod from the rubber tubing and connect 
it with the thistle-tube funnel. Fill the funnel with Ringer's 
solution and open the lateral tube of the cannula to allow any 
air bubbles that may be in the tubing to escape. The fluid, 
entering the aorta, makes the complete circuit of the frog's 
vessels, and returns to the sinus venosus, where it escapes, 
and, running down the frog's legs, drops from its toes into a 
pan placed beneath them. Let the perfusion continue until 
the fluid dropping from the toes is fairly free from blood. 
1. Now count the number of drops falling from the toes 
in two periods of two minutes each, and record the results 
in a protocol as the normal. 
2. Empty the funnel through the side tube, and refill it 
with a solution of adrenalin in Ringer's sol. Let the fluid 
perfuse for a few minutes (until the flow is markedly dimin- 
ished) and count the number of drops in two periods of two 
minutes each. Record your results as before. 
3. Empty the funnel, and refill it with a solution of amyl 
nitrite in Ringer's sol. Let the fluid perfuse for a few minutes 
(until the number of drops from the toes show marked in- 
crease). Count the number of drops in two periods of two 
minutes each. Record the results. 
4. Empty the funnel and perfuse again with Ringer's sol. 
Count the number of drops for two periods of two minutes 
each for the second normal. 
5. Now destroy the spinal cord and count the number of 212 
Laboratory Exercises in Physiology 
drops for two periods of two minutes each. Record the re- 
sults. 
6. Perfuse again with adrenalin solution and record the 
number of drops in two periods of two minutes each. 
7. Empty the funnel and refill it with amyl nitrite solution. 
Count the number of drops in two periods of two minutes 
each. The effects of these drugs are shown here not partic- 
ularly to demonstrate their action, but tq show how constric- 
tion and dilatation of the blood-vessels influence the flow of 
fluids through them. 
Observe that adrenalin causes a marked decrease in the 
flow by constricting the vessels, both before and after de- 
stroying the spinal cord. Does it produce this effect by its 
action on the nerve centers or by direct action upon the vessel 
walls? Observe that amyl nitrite causes a decided increase in 
the flow by dilating the blood-vessels, both before and after 
destroying the cord. How does it produce the dilatation? 
Observe that destroying the spinal cord greatly increases the 
flow of Ringer's sol. through the vessels, because destruction 
of the vasomotor centers in the cord removes vasomotor tone. 
ARTERIAL PRESSURE IN MAN. 
141. The Sphygmomanometer: A number of instruments 
have been devised in recent years for measuring blood-pressure 
in man. The principle is practically the same in a number 
of them. The one designed by Erlanger gives accurate results 
by the graphic method. 
On the inside of a rigid leather armlet or cuff is a broad, 
flat rubber bag which can be inflated with air so as to com- 
press the brachial artery. The armlet is buckled about the arm 
so that the rubber bag comes over the brachial artery; the 
rubber bag is connected, by means of a three-way stop cock, 
(a) with a rubber bulb for inflating the bag, (b) with a mer- 
cury manometer for registering the pressure and (c) with a 
thick-walled rubber bulb enclosed in a glass chamber. The 
pulsations in this bulb are transmitted by the air in the glass 
chamber to a tambour; the lever of the tambour magnifies 
and records the pulsations on a revolving drum. 214 
Laboratory Exercises in Physiology 
Owing to the compressibility and elasticity of air, the pres- 
sure within the arm-piece is the same as in the other parts of 
the apparatus, and it is measured by the mercury manometer. 
The pressure which is just sufficient to obliterate the radial 
pulse corresponds to the maximal or systolic pressure in the 
artery. With most instruments this point is determined by 
feeling the radial pulse at the wrist. With the Erlanger ap- 
paratus this point may be seen on the tracing by the 
sudden increase in the height of the pulsations when the pressure 
is lowered, after having been above the pressure required to 
obliterate the artery. This point shows the pressure at which 
the blood just begins to get through when the compression 
is lessened, and is usually from 5 to 10 mm. of mercury lower 
than the point at which the radial pulse disappears at the 
wrist. 
The diastolic pressure, or the pressure between the beats of the 
heart, corresponds to the pressure at which the greatest pulsa- 
tions occur. It has been found by experiment that the greatest 
lateral expansion of the artery with each heart beat, occurs 
when the pressure on the outside of the artery is just equal 
to the diastolic pressure of the blood within the artery. 
With the Janeway and Stanton sphygmomanometers (which 
consists of a broad arm-piece and a mercury manometer with 
narrow lumen) this point is determined by observing the pres- 
sure at which the greatest pulsations of the mercury occur. 
With the Erlanger apparatus the last high, waves on the tracing, 
^s the air is allowed to escape slowly, mark the diastolic pressure. 
The mean arterial pressure is determined by finding the mean 
between the systolic and diastolic pressure. 
Each student will in turn determine the blood pressure in 
man (a) with the Erlanger sphygmomanometer; (b) with a 
modified Erlanger apparatus; (c) with a modified Stanton or 
Janeway apparatus; (d) with the Faught sphygmomanometer, 
and (e) by the auscultatory method. 
The individual whose pressure is to be determined is seated 
near the table with the forearm resting on it, at the same 
level as the heart. The arm-piece is fastened snugly, but not 
too tightly, about the arm just above the elbow. Care should 
be taken that the rubber bag is over the line of the brachial 216 
Laboratory Exercises in Physiology 
artery. The arm-piece is inflated until the pulse at the wrist 
is just obliterated. The pressure at this point should be noted. 
The side tube is now opened and air allowed to escape 
through a fine capillary tube until the pulse is perceptible at 
the wrist. The pressure at this point is also noted and the 
mean between the two may be taken as the systolic pressure. 
The observation should be repeated several times until uniform 
results are obtained. 
Now inflate the arm-piece until the pressure in the arm-piece 
is above the systolic pressure; allow the air to escape slowly 
through the side opening, watching carefully the pulsations 
in the mercury manometer; observe the pressure at which the 
highest pulsations occur. This is the diastolic pressure. Re- 
peat the observation several times. 
Auscultation Method: Inflate the pneumatic bag as before 
until the artery is obliterated. A stethoscope is applied over the 
artery below the cuff and the pressure within the bag is allowed 
to fall gradually. A distinct sound or sharp thump is heard at 
the point at which the blood gets past the compression and the 
circulation commences. This marks the systolic pressure. 
The sound undergoes a number of changes as the pressure in 
the pneumatic bag is lowered. Just before it disappears it 
change from a loud to a dull, muffled sound. This corresponds 
to the diastolic pressure. The sound completely disappears when 
the pressure in the bag falls a few millimeters below the diastolic 
pressure. 
Repeat these observations several times until uniform results 
are obtained. Record the results by the different mthods and 
compare them. 
Erlanger gives the average systolic pressure at 110 mm. and 
the average diastolic pressure as 65 mm. for young adults 
under 25 years of age. The average obtained in this laboratory 
upon fifty healthy medical students is 127 mm. Hg for the 
systolic and 85 mm. Hg for the diastolic pressure. With the 
Janeway apparatus the figures are usually from 5 to 10 mm. 
higher than those obtained with Erlanger's sphygmomanometer. 
142. The Blood-Pressure in the Capillaries: This can not 
be measured directly, but is determined by the pressure re- 218 
Laboratory Exercises in Physiology 
quired to obliterate the capillaries in compressing a thin layer 
of soft vascular tissue. 
(a) See that the capillaries of the hand are filled (by hold- 
ing it down or by placing it in warm water for a short time). 
Place the little glass square of the special weight pan on the 
skin just back of the finger nail, holding the hand at the level 
of the heart. This glass is % cm. square, and therefore has 
an area of sq. cm. Add weights to the pan until the pink 
color of the skin disappears. This marks the point at which 
the capillaries are obliterated. Add the weight of the pan 
to the weights in it and divide by 1.359. The result will ex- 
press the pressure in mm. of mercury as a vertical column of 
mercury 1 mm. high and Vi SQ- cm. section exerts a pressure 
of 1.359 grams. Record the result of your observations. 
(b) Place the finger on the pneumatic cushion made by a 
finger cot in a curved metal support. Inflate the finger cot 
until it presses the finger against the glass across the top of 
the frame with sufficient force to obliterate the blood vessels. 
The pressure is indicated by a mercury manometer connected 
with a T-tube between the inflating bulb and the finger cot. 
143. Tracing of the Pulse Wave: Smoke the drum lightly 
and arrange the recording tambour to write with the least 
friction possible. Connect the tambour with a thistle tube 
by heavy tubing, with a T-tube interposed between the two. 
See that the side arm of the T-tube is open. Use the thistle 
tube as a receiving tambour (without a membrane) by pressing 
backward and inward over the carotid artery, just in front 
of the sterno-cleido-mastoid muscle, about the level of the 
thyroid cartilage. (The point of the best pulsation can readily 
be determined by first pressing with the finger.) When all is 
ready have the assistant close the side arm of the T-tube with a 
spring pinchcock and set the drum at slow speed. If good pulsa- 
tions are not obtained at first, the pressure with the thistle 
tube should be varied until good pulsations are registered on 
the drum. The arm and hand holding the thistle tube should 
be held steady, resting on the table in a comfortable position. 
Take one tracing with the drum at such speed that each 
wave occupies about 1 cm. on the tracing, and then take an- 220 
Laboratory Exercises in Physiology 
other with the drum at fast speed. With the electro-magnet 
record seconds under each tracing with the drum at the same 
speed as before. 
Observe (a) the percussion cap; (b) the dicrotic notch and 
dicrotic wave; (c) the predicrotic wave which may not be 
constant. 
144. Sphymogram with the Dudgeon Sphygmograph: With 
the finger first determine the position at the wrist where the 
pulsation of your partner's radial artery is greatest. Adjust 
the Dudgeon sphygmograph so that the pad comes exactly 
over this point and hold the instrument securely by the tapes 
passed about the wrist. See that the clock work is wound; 
vary the pressure of the spring and pad over the artery until 
the greatest excursions of the recording pen are obtained; 
then take a tracing. Varnish the tracing and paste it in 
the book with a brief description of each part. 
145. Cardiogram: Smoke the drum lightly and arrange a 
recording tambour as before. Connect it with a small funnel 
by rubber tubing with a T-tube between the two. 
Adjust the small funnel which acts as a receiving tambour 
over the apex beat or cardiac impulse and hold it steady while 
your partner takes the tracing. The best results are obtained 
by leaning forward and to the left. 
Take one tracing with the drum at moderate speed and an- 
other with the drum at fast speed. With the electro-magnet 
record seconds beneath each tracing with the drum at the 
same speed as before. 
Observe the small wave which precedes the main wave. The 
former corresponds to the systole of the auricles and is caused 
by the complete filing of the ventricles at that phase of the 
heart cycle; the latter marks the systole of the ventricles and 
is due1 to the hardening of the heart muscle which comes in 
closer apposition with the chest wall during contraction. 
The best pulsations are obtained between respirations by 
holding the breath after a deep expiration. The respiratory 
movements of the chest and the expansion of the lung interfere 
with uniformity in the tracing. 
If good cardiograms can not be obtained at first, a little 
active exercise will cause a stronger cardiac impulse. 222 
Laboratory Exercises in Physiology 
METABOLISM. 
ABSORPTION FROM THE AILMENTARY CANAL. 
146. Seat of Absorption: A fasting dog or cat is anes- 
thetized and a small opening is made in the abdominal wall 
to expose the stomach and small intestine. If the intestine 
contains any food it is emptied by stripping between the fin- 
gers. Two ligatures are tied aronnd the intestine about three 
feet apart; ligatures are also tied about the cardiac and pyloric 
orifices of the empty stomach. 
The same amount of water is then injected into the stomach 
and into the loop of the intestine (50 c.c. for a cat; 75-100 
c.c. for a dog). The injection is made by means of a hypo- 
dermic needle passed obliquely through the wall. Some blood 
serum may be injected into another loop of the small intestine. 
After 60 to 75 minutes the animal is killed and the water 
that remains in the stomach and in the loop of the intestine is 
collected separately and measured. Observe (1) that rela- 
tively little of the water has been absorbed from the stomach, 
although its wall is as vascular as that of the intestine; (2) 
that most or all of the water has been absorbed from the 
intestine; (3) that blood serum is absorbed from the intestine 
although it has the same osmotic pressure as the blood and 
the capillary blood pressure is much greater than the pressure 
in the lumen of the intestine. Record the results. 
147. Absorption of Colloids and Crystalloids from the 
Intestine: A fasting dog is anesthetized and the small in- 
testine is exposed. It is emptied of any food that may re- 
main by stripping the jejunum and ileum between the fingers 
from above downward. The intestine is then divided into 
four separate loops, each about 15 inches long, with ligatures 
tied lightly about the upper and lower ends. Each loop has 
two ligatures, with 1 or 2 cm. of intestine intervening between 
the loops, so that each one can be removed separately later and 
its contents collected for measurement. 
With a large syringe and a fine hypodermic needle 20-25 c.c. 224 
Laboratory Exercises in Physiology 
the following solutions are injected into the lumen of the dif- 
ferent loops from above downward : 
(a) 10 per cent sol. of egg white. 
(b) 2 per cent sol. of commercial peptone. 
(c) 2 per cent sol. of dextrose. 
(d) 2 per cent sol. of potassium iodide or a 0.25 per cent 
sol. of sodium sulphate. 
The intestine is carefully returned to the abdominal cavity 
with as little injury as possible and the blood supply of the 
intestine is not disturbed. 
At the end of one and one-half hours the animal is killed 
and the loop removed. The fluid in each loop is collected sep- 
arately and measured. 
Observe (1) that there has been complete absorption of the 
peptone; (2) that a large part but not all of the egg albumin 
solution has been absorbed; (3) that the loop into which dex- 
trose had been injected is entirely empty as there has been 
complete absorption; (4) that the potassium iodide or sodium 
sulphate shows comparatively little absorption, the greater 
part remaining in the loop into which it was injected, although 
it is as diffusible as dextrose. 
Quantitatively the amount of albumin, potassium iodide, or 
sulphate remaining unabsorbed in the intestine can be deter- 
mined. It corresponds practically to the proportion of the so- 
lution which remained in the loop unabsorbed. 
Record the result and explain the absorption of the colloidal 
and non-diffusible egg albumin. 
Explain why one crystalloid (dextrose) is completely ab- 
sorbed and the other only slightly. 
Can absorption from the living intestine be explained by 
known physical law's? 
How does absorption from the intestine differ from absortion 
from the tissues and from the serous cavities of the body. 
148. Absorption from the Large Intestine: This may be 
shown in the dog by the rectal injection of 75 per cent ether 
in oil after the colon has been emptied. When it is retained 
there is sufficient absorption to produce narcosis. 
Absorption of ether vapor takes place to a sufficient extent to 226 
Laboratory Exercises in Physiology 
produce anesthesia in a rabbit when a catheter passed into the 
rectum is connected with a flask containing ether and immersed 
in a basin of warm water to promote vaporization. 
149. Glycogen in the Liver: A rabbit is fed upon a liberal 
allowance of starchy food for a day before the experiment. The 
animal is killed by bleeding and the liver quickly removed. It is 
chopped into small pieces; a part of the liver is weighed and 
quickly thrown into a definite amount of boiling water made 
slightly acid by the addition of a few drops of acetic acid. This 
kills the liver cells, coagulates the proteids and destroys any 
enzymes which might convert the glycogen into dextrose. 
After boiling for a time, the liver is ground up in a mortar 
with washed sand, boiled again and filtered while hot. The 
nitrogenous bodies which have not been coagulated by heat are 
then removed from the filtrate by Brucke's method (i. e., adding 
cone. HC1 and potassio-mercuric iodide sol. alternately until no 
more precipitate forms). The precipitate is filtered off and the 
filtrate is comparatively a pure sol. of glycogen. Distilled water 
is added to the filtrate to bring it up to a definite volume and 
restore the water lost by boiling. 
(1) Note the opalescence of the solution which is character- 
istic of glycogen. (2) Observe the deep reddish-brown color 
from the addition of a few drops of a weak iodine solution. (3) 
The glycogen may be precipitated by adding an excess of alcohol. 
Pfliiger's method of determining the amount of glycogen in 
tissues insures more complete extraction than Brucke's method, 
but it requires a longer time and for that reason can not be used 
for the purposes of the present and subsequent experiments. 
It is based on the complete digestion and solution of the tissue 
by boiling in 60 per cent KOH for three hours. The glycogen 
is then precipitated by adding alcohol in such amount that all 
of the glycogen but none of the protein matter is precipitated. 
By repeated washings and precipitation the glycogen is obtained 
in pure state. 
150. Glycogen in Muscles: An equal quantity (by weight) 
of the voluntary muscles of the same rabbit is chopped up and 
extracted in the same way in the same volume of water. After 
removal of the nitrogenous bodies, water is added to restore that 228 
Laboratory Exercises in Physiology 
lost by boiling and bring the solution np to a definite volume as 
before. Comparison may be made between the two extracts as 
to the amount of glycogen present. 
Note that muscles contain glycogen and the extracts respond 
to the tests given above. How does it compare in amount with 
the quantity of glycogen in the extract of the liver? Record 
the result. 
151. Disappearance of Glycogen in the Liver: A part of 
the rabbit's liver is kept moist in the incubator at bodily temper- 
ature for an hour or more. A definite amount, by weight, is 
then extracted in a proportionate volume of water in the same 
way as before. This should be compared with the extract of 
the first portion of liver. 
Observe that the amount of glycogen is much less. This may 
be determined (1) by the degree of opalescence; (2) by the 
depth of color with iodine in comparative tests; (3) by the pre- 
cipitate with alcohol. Record the results. 
Observe also that the amount of sugar in the second portion of 
liver is greater than that in the first portion. This may be de- 
termined by the amount of precipitate formed by boiling a 
definite amount of diluted Fehling's sol. with the same amounts 
of the liver extracts in comparative tests. Record the result. 
From what you have seen, write a brief account of the relative 
amount of glycogen stored in the liver and muscles and the 
change that takes place in it after death. 
152. Ability of the Blood to Handle Dextrose; Glycosuria 
from Hyperglycemia: A dog is prepared for making intra- 
venous injections by the jugular vein; the urine is collected from 
the ureters. Some normal urine is collected and examined to 
see that it is free from sugar. 
The amount of blood, and the quantity of dextrose in solution 
in the blood, are calculated from the weight of the animal. 
This may be based upon the average normal amount (0.1 per 
cent) or the maximum amount normally present (0.15 per cent). 
A sufficient amount of dextrose is then injected to increase 
the quantity of sugar in the blood first by 50 pef cent, and later 
by 100 per cent. The urine is then collected for periods of five 
minutes each after each injection. Note that the urine remains 230 
Laboratory Exercises in Physiology 
free from sugar until the percentage of sugar in the blood ex- 
ceeds 0.2 per cent, or in exceptional instances 0.3 per cent. 
Observe that glycosuria does not occur while there is less than 
0.2 per cent of dextrose in the blood. Make a protocol and 
record the result of the experiment. 
153. Effect of Injecting Maltose, Blood Serum and Foreign 
Proteins: 1. Another dog is prepared in the same way. The 
normal urine is collected and examined to be sure that it is 
free from sugar and albumin. A small amount of maltose (0.1 
per cent of the blood or less) is injected intravenously and the 
urine collected. Observe that maltosuria occurs within a few 
minutes, (a) Can maltose be utilized when injected intra- 
venously? (b) What change takes place in maltose as it is 
absorbed from the intestine? 
2. A definite amount of blood serum is injected intravenously. 
Observe that albuminuria does not result. 
4. A solution of egg albumin, containing approximately the 
same amount of coagulable protein as the blood serum, is in- 
jected intravenously. Observe that albuminuria soon follows. 
Can egg albumin be assimilated and utilized when injected di- 
rectly into the blood, or is it eliminated as a foreign body? 
154. Glycolytic Action of Shed Blood: 1. A definite amount 
(10 grams) of fresh defibrinated dog's blood is mixed with an 
equal amount (by weight) of a saturated Na2SO4 solution and 
acidulated with acetic acid. This is boiled until the foam is 
white and distilled water added to make 20 grams. This is then 
filtered and the filtrate is gradually added from a burette to a 
definite amount of Fehling's or Purdy's sol. while the latter is 
boiled in an evaporating dish. Enough of the filtrate is added 
to cause the blue color to completely disappear. The amount 
of sugar, as indicated by the quantitative method given above, 
may be taken as the normal for comparison with the following: 
2. Defibrinated blood that has been standing for three or four 
hours after removal from the same animal, treated in the same 
way, shows less sugar than fresh blood. 
3. Blood from the same animal that has been standing for 
eighteen or twenty-four hours shows very little or no sugar when 
treated in the same way. Record the results. 232 
Laboratory Exercises in Physiology 
155. Puncture Diabetes: A rabbit has been fed upon a 
liberal allowance of carbohydrates for a day or two before the 
experiment. The normal urine is collected before the operation 
and examined for sugar. 
The floor of the fourth ventricle (Bernard's so-called diabetic 
center) is punctured with a blunt probe. After an hour or two 
the urine is expressed from the bladder and shows pronounced 
glycosuria. 
Write a brief account of this form of glycosuria, telling how it 
is produced, the conditions necessary for its production, the in- 
fluence of vasodilatation in other parts, and whether or not 
hyperglycemia is present. 
156. Phloridzin Diabetes: The urine of a dog is collected 
and examined for sugar. Phloridzin is then given (2 or 3 
grams) either by the stomach or hypodermatically. Several 
hours later the urine shows an abundance of sugar when exam- 
ined by Fehling's solution, Barfoed's reagent, or the phenyl- 
hydrazine test. 
The sugar content of the blood is also determined. The pro- 
teids of blood must be removed before the quantity of sugar can 
be determined by its reducing power. This may be done as in- 
dicated above, or by the following method: Fresh blood is boiled 
with five times its weight of a mixture of phosphotungistic acid, 
70 grams, and HC1 (Sp. Gr. 1.20) 20 c. c., in a liter. It is then 
filtered, neutralized and diluted to a definite valume. 
Write a brief account of this form of glycosuria, telling how it 
is produced, how long it lasts, and whether the amount of sugar 
in the blood is changed or not. Does the amount of sugar given 
off in the urine in this form of diabetes depend upon the amount 
of glycogen stored in the tissues'? 
157. Pancreatic Diabetes: This may be produced by re- 
moving seven-eighths or more of the pancreas. All the phe- 
nomena of true diabetes (glycosuria, polyuria, emaciation, in- 
tense thirst, acidosis, etc.) occur in depancreatized dogs that 
survive the operation for some time. Tn pancreatic diabetes 
there is a condition of hyperglycemia. 
158. Adrenalin Glycosuria: The hypodermatic injection of 234 
Laboratory Exercises in Physiology 
adrenalin chloride (1-3 c. c. of 1-1000 sol. diluted with physi- 
ological salt sol.) causes a transitory glycosuria which only lasts 
a few hours. 
159. The Nervous Mechanism of Salivary (Submaxillary) 
Secretion: The submaxillary gland of a large dog is exposed 
and a cannula is tied in its duct; the chorda tympani nerve and 
the sympathetic fibers about the artery are also exposed so that 
they may be stimulated. Count the number of drops for two 
normal periods of one or two minutes each. 
1. Reflex Stimulation: Some diluted acetic acid is applied 
to the mucous membrane of the mouth. Observe the increased 
flow of saliva, counting the number of drops for two periods 
and recording the result in a protocol of the experiment. 
Later in the experiment the chorda tympani will be cut. Note 
that acetic acid, applietl to the mouth as before, fails to excite 
any flow from the submaxillary gland after section of the chorda 
tympani, because the "reflex arc" is broken. Record the result 
of this observation. 
2. The Chorda Tympani Nerve: (a) Observe the effect of 
stimulating the chorda (1) on the flow of saliva, and (2) on the 
vascularity of the gland. Record the results of stimulation in 
the protocol. 
(b) What change in the character of the secretion as deter- 
mined by the viscidity ? 
(c) What effect does cutting the chorda tympani have upon 
the flow of saliva? 
(d) What results from stimulating the peripheral end of the 
nerve ? Record the result. 
(e) What kinds of nerve fibers go to the submaxillary gland 
in the chorda tympani? Are these afferent or efferent? 
3. Sympathetic Fibers to the Submaxillary Gland: What is 
the effect of stimulating these fibers upon (a) the amount and 
(b) the character of the secretion? What change takes place in 
the state of the blood-vessels from stimulation of the sympa- 
thetic? What are the functions of the sympathetic fibers to the 
submaxillary gland? 
4. Effects of Pilocarpine and Atropine: (a) Observe and 
record the effect of 0.001-.002 gram, pilocarpine given intraven- 236 
Laboratory Exercises in Physiology 
ously, upon the secretions of the submaxillary gland. Count the 
number of drops for two periods of two minutes each. 
(b) Observe the effect of atropine given intravenously in the 
same dose and record the result. 
(c) Can the secretion be increased by stimulating the peri- 
pheral end of the chorda tympani after giving atropine? Is the 
vascularity increased? Would you conclude from this, that the 
vasodilatation alone was responsible for the increased flow of 
saliva when the chorda was stimulated before the administration 
of atropine? 
(d) Stimulate the sympathetic nerve trunk after the admin- 
istration of atropine and record the result. Are the sympathetic 
fibers paralyzed by atropine as completely as the secretory 
fibers of the chorda tympani? 
(e) Does a second dose of pilocarpine have any effect after 
the secretion has been stopped by atropine? What is the action 
of pilocarpine and atropine, respectively, upon the secretory 
activity of the salivary glands? 
160. Histological Changes in Secreting Glands: These 
changes are best seen in fresh, teased specimens, as fixation agents 
cause changes in the appearance of the cytoplasm and granules 
within the gland cells. A rabbit is starved for twenty-four hours 
so that there is an abundance of zymogen granules in the gland 
cells. This gives a true picture of the gland in the resting stage. 
1. The animal is lightly anesthetized and a small piece of the 
parotid gland cut off. This is teased on slides in the rabbit's 
saliva. Examine the specimen with the low and also with the 
high power of the microscope. Describe the appearance of the 
resting gland and draw a picture of it. Note that the lobules of 
the gland are distended, the cells being filled with coarse proto- 
plasmic granules which obscure the nuclei and make the outlines 
of the cells indistinct. 
2. Secretion is produced by injecting small doses of pilocar- 
pine from time to time, so as to cause a copious flow of saliva. 
Small bits of the parotid are clipped off and teased in saliva as 
before. Examine and describe the appearance of pieces of the 
gland taken after one hour, one and one-half hours and two hours 
of very active secretion. Draw pictures of the later stages to 238 
Laboratory Exercises in Physiology 
show the appearance of the "discharged" gland. Note that the 
zymogen grannies are much less abundant and are most numer- 
ous toward the middle of the lobule; that the nuclei and outlines 
of the cells are more distinct. 
161. Histology of the Thyroids and Parathyroids: Review 
the normal histology of these glands. Observe the closed acini 
of the thyroid, lined by low cubical epithelium and filled with 
colloid which stains well with eosin. 
The epithelial cells of the parathyroids have relatively less 
protoplasm and the general arrangement is totally different. The 
cells are arranged in anastomosing columns in some places; in 
other parts there is no definite arrangement, the cells being 
closely packed together with an irregular distribution of vascu- 
lar connective tissue. Note that there are no distinct acini and 
that there is no colloid. 
Study the sections of an hypertrophied external parathyroid, 
which remained for several weeks in a dog after the removal of 
the thyroids (with the internal parathyroids). Observe that 
distinct acini have developed throughout the gland and the epi- 
thelial character of the cells is more apparent. While a colorless, 
homogenous substance is present in the lumen of some of the 
acini, there is no evidence that this is colloid. The appearance 
is that of hypertrophy, rather than that of a transformation of 
parathyroid to thyroid structure. 
162. Thyroidectomy: Both thyroids, with the internal par- 
athyroids, have been removed from a dog, leaving the external 
parathyroids intact with their circulation undisturbed. 
Observe the condition of this animal from time to time for 
several weeks after the operation and write a brief account of 
the results of the experiment. Note that thyroidectomy (with- 
out parathyroidectomy) does not produce any symptoms in dogs. 
163. Parathyroidectomy: All four parathyroids are re- 
moved from a dog with the least possible injury to the thyroids. 
The circulation to the thyroids is not disturbed. 
Observe that the animal becomes seriously ill in a very short 
time. Anorexia is present from the first; tetany begins in a few 
days, showing itself as fibrillary contractions of the muscles, 240 
Laboratory Exercises in Physiology 
especially seen in the tongue; as the tetany grows worse there 
are muscular twitchings and local spasms; there may be rigidity 
of the muscles and ultimately general convulsions. At times 
polypnea is present. Death invariably ensues in a variable time 
(usually two days to two weeks). 
These symptoms are the same as those which follow "total 
thyroidectomy" in dogs, i. e., complete excision of both thyroids 
and all four parathyroids. Formerly these symptoms were at- 
tributed to the removal of the thyroids. From the experiments 
that you have seen, which set of bodies would you consider the 
more essential to life? What causes the tetany and death when 
both sets of bodies are removed? 
Write a brief account of the results of the parathyroidectomy 
and compare it with a third dog in which both sets of bodies are 
removed. 
164. Influence of Transfusion on Parathyroid Tetany: The 
transfusion of normal blood to a dog with parathyroid tetany 
causes a transitory disappearance of the nervous symptoms, but 
the blood of the dog with tetany does not have any effect when 
trnsfused into a normal dog. 
165. Influence of Calcium on Parathyroid Tetany: Observe 
the effect of giving full doses of calcium acetate or calcium lac- 
tate to a dog suffering from severe tetany following parathyroid- 
ectomy. Does it modify the severity of the nervous symptoms 
or cause them to disappear? Does it prolong the life of the ani- 
mal? Write an account of the experiment. 
EXPERIMENTS ON THE KIDNEY. 
166. Pressure in the Ureter: The ureters are exposed 
through lumbar incisions and cannulas are tied in them. One of 
the ureters is connected to a mercury manometer with a T- 
tube interposed between the two. Physiological salt solution is 
injected into the ureter by means of the side arm of the T-tube 
until the pressure in the manometer begins to rise. Observe and 
record the ureter pressure which is sufficient to stop the further 
formation of urine. How does this pressure compare with the 
capillary blood-pressure ? 242 
Laboratory Exercises in Physiology 
167. Saline Diuretics. (1) Na Cl Diuresis: After the flow 
of urine returns to the normal, a hypertonic solution of NaCl 
(1.5 per cent solution) is injected intravenously. Observe the 
increased flow of urine which lasts for a little time. The urine 
is collected for five-minute periods as before. Record the re- 
sults. Observe the change in the character of the urine. This 
may be determined by taking the specific gravity and also by 
determining the amount of urea given oft' for each period. 
Record the results and compare them with the normal urine 
collected at the beginning of the experiment. 
Observe that the urine formed during NaCl diuresis contains 
dextrose. It has been found by experiment that there is no 
hyperglycemia as the result of injecting NaCl solution. How 
do you explain the NaCl glycosuria? 
Sodium sulphate when injected intravenously causes a greater 
diuresis than sodium chloride. 
168. (2) Dextrose Diuresis: After the flow of urine re- 
turns to the normal, a concentrated solution of dextrose (10 
to 20 per cent solution) is injected. Observe the tremendous 
increase in the flow of urine. Record the results for five minute 
periods. Note that the specific gravity is lowered and the 
output of urea for the five-minute periods is diminshed as 
before. 
It has been found that a preliminary bleeding to the extent 
of the amount of water attracted into the blood by the osmotic 
pressure of the dextrose injected, prevents the diuresis to a 
large extent. 
From this it appears that crystalloid diuretics act as such, 
to a large extent, by producing hydremic plethora and the 
consequent increase in the capillary blood-pressure. The NaCl 
glycosuria indicates that the permeability of the renal epithelium 
is also changed. 
169. Influence of Changes in the Circulation Upon the For- 
mation of Uurine: The normal urine is collected for two or 
three periods of five minutes each. Make a protocol of the 
experiment, recording the quantity of urine, the specific grav- 
ity and the urea content for each of these periods. The blood- 
pressure is taken in the carotid artery. 244 
Laboratory Exercises in Physiology 
1. Adrenalin is then injected intravenously in small quanti- 
ties to keep the blood-pressure elevated above the normal. 
Observe that the excretion of urine is decreased, in spite of 
the increased blood-pressure, because the renal arterioles are 
constricted. Record the result. 
2. After the flow of urine returns to the normal, alcohol, 
properly diluted, may be injected. The flow of urine is in- 
creased, although the general blood-pressure is not increased, 
and may even be lowered. The increased formation of urine 
may be explained by the vasodilatation and the increased ve- 
locity of the blood stream (acceleration of the heart). Record 
the results in the protocol. 
170. Influence of the Splanchnic Nerve on the Renal Ves- 
sels and Flow of Urine: The number of drops of urine falling 
from the cannula in each ureter is counted for several periods 
of one minute each. Record the results. 
The left splanchnic nerve is then cut just above the dia- 
phragm. Count the drops from each ureter as before and re- 
cord the result. Note the increase in the flow from the left 
kidney and explain the cause of it. 
The peripheral end of the cut splanchnic is then stimulated. 
During the stimulation count the drops from each ureter as 
before and record the result. How do local vasoconstriction 
and local vasodilatation influence the flow of urine? Make a 
complete protocol of the experiment. 
171. Influence of Lowered Blood-pressure Upon the Ex- 
cretion of Urine: Record the amount of urine excreted from 
both kidneys in a five-minute period. A transverse section of the 
spinal cord is made in the upper cervical region to lower the 
arterial blood-pressure. Artificial respiration is maintained. 
Observe whether or not any urine is formed after the mean 
arterial pressure (measured in the carotid) falls below 40 
mm. Hg. Record the residt and compare with the formation 
of urine before cutting the spinal cord. What arterial pres- 
sure is necessary for the formation of urine? 
172. Specific Diuretics: The normal urine of another dog 
is collected for several periods of five minutes each; the specific 246 
Laboratory Exercises in Physiology 
gravity is taken and the output of urea for each period is de- 
termined. 
Caffeine or diuretin (theobromine salicylate) is injected in- 
travenously. The urine is again collected for three periods 
of five minutes each; the specific gravity and urea output are 
determined as before. Record the results in a protocol of the 
experiment. Observe (a) that the quantity of urine is in- 
creased; (b) the specific gravity is lowered; (c) the urea out- 
put is increased. How does this compare with the urine ex- 
creted during saline diuresis? 
The diuretic action of caffeine or diuretin is much more pro- 
nounced on rabbits than on dogs. 
Do the specific diuretics seem to act upon the kidney by 
changing the physical forces concerned in the formation of 
urine, or by some specific action upon the renal epithelium? 
173. Urea as a Diuretic: A solution of urea is injected in- 
travenously after the flow of urine returns to the normal. 
Observe that the flow of urine is greatly increased; the specific 
gravity is lowered. Record the results. Observe that urea 
does not show any toxic effects, even when injected in large 
quantity. It seems to act as a crystalloid diuretic by pro- 
ducing hydremic plethora. Possibly the permeability of the 
renal epithelium is also altered as glycosuria often follows the 
injection of urea as it does after the injection of NaCl. 
174. Compression of the Blood-vessels of the Kidneys: The 
kindney is exposed on one side and the renal vein is separated 
from the other vessels. The renal vein is compressed between 
the fingers for one minute. Observe that the flow of urine 
stops. 
The other kidney is exposed in the same way and the renal 
artery is compressed between the fingers for one minute. Ob- 
serve that the flow of urine stops for a considerable period. 
MUSCLE AND NERVE. 
175. The Nerve-Muscle Preparation: Pith a frog and de- 
stroy the brain and eord. Wrap a towel around the hind legs 
and hold them in the left hand, the ventral surface being down. 248 
Laboratory Exercises in Physiology 
With stout scissors transfix the body at about the fifth or sixth 
vertebra and divide the spinal column. The viscera, together with 
the anterior and ventral parts of the body, fall away from the 
pelvis and the dorsal part. Now remove the viscera by cutting 
the adbdominal walls with scissors, parallel to the vertebral col- 
umn and close to the pelvis. Strip the skin down over the lower 
end of the trunk; with scissors cut through the skin about tilt 
anus; now continue to pull the skin until the posterior extremi- 
ties are completely denuded. 
Holding the thigh muscles between the thumb and the fingers, 
carefully tear the fascia between the biceps and semi-membran- 
osus muscle with a seeker and separate the deeper muscles until 
the sciatic nerve and the femoral vessels come into view. Clear 
the nerve with a seeker, or a glass rod drawn to a smooth point, 
taking care not to stretch or injure it in any way. (The nerve 
should not be handled with forceps.) 
Divide the pyriformis and ilio-coccygeus muscles and free the 
nerve up to the last vertebra by lifting the tip of the urostyle 
(coccyx) and cutting it away. A branch of the sciatic nerve 
coming off at the upper part of the thigh must be divided with 
the scissors. Now cut the sciatic nerve as high up as possible. 
Observe that the muscles supplied by the nerve contract when 
the nerve is cut. 
Separate the tendo-Achillis and sever it below the fibro-carti- 
lage. Lift the tendon and free the gastrocnemius as far up as 
the lower end of the femur. With stout scissors cut across the 
knee joint, removing the tibio-fibula with its muscles and the 
foot. Now lay the nerve on the gastrocnemius and cutting 
through the muscles of the thigh, divide the femur about its 
middle. The nerve and muscle should be kept moist with physi- 
ological salt solution and the nerve should never be allowed to 
touch the skin. If the preparation is not to be used at once it 
should be wrapped in filter paper moistened with physiological 
salt solution and must not be allowed to dry. 
Arrange the preparation in the moist chamber by fastening 
the end of the femur in the clamp; lay the nerve on the plati- 
num electrodes, handling it with a camel hair brush moistened 
with physiological salt solution. The tendo-Achillis should be 250 
Laboratory Exercises in Physiology 
attached to the muscle lever by means of an S-shaped hook passed 
through the tendon above the sesamoid bone. Attach a 20-gram 
weight to the wire which lies in the groove of the axis-wheel of 
the lever, by means of a wire around the neck of the weight, 
and adjust the screw beneath the muscle lever so that it supports 
the weight until the muscle is taut but not stretched. A muscle 
that is thus loaded only during contraction and not kept on a 
stretch while at rest, is said to be "after loaded." 
176. Different Forms of Stimulation: (a) Mechanical 
Stimulation may be applied by pinching the free end of the 
nerve with forceps. Observe that the contraction results just 
as it does after cutting the nerve or its branches. 
(b) Chemical Stimulation may be produced by dipping the 
end of the nerve in glycerine or a concentrated solution of sodi- 
um chloride. A chemical stimulus should only be applied in a 
watch crystal to the free end of the nerve and the part of the 
nerve affected should be cut off with the scissors. 
(c) Electrical Stimulation may be applied in the form of 
make and break induction shocks or by the make and break of 
the constant (galvanic) current. 
(1) Single Induction Shocks. Minimal, Submaximal and 
Maximal Contractions: Arrange the inductorium for single in- 
duction shocks with a simple key in the primary circuit; connect 
the binding posts of the secondary coil with those of the elec- 
trodes in the moist chamber. 
Start with a very weak current which can not be felt; grad- 
ually change the position of the secondary coil until the induc- 
tion shocks at the break of the current in the primary begin to 
be felt. Note the position of the secondary coil and record the 
height of the contractions on the drum while it is standing still. 
Move the drum a short distance by hand after each contrac- 
tion and take a series of tracings to show the increase in the 
height of the contractions as the strength of the stimulus is grad- 
ually increased. Mark the contractions that occur at the make 
(m) and at the break (b) of the current in the primary. In- 
crease the strength of the stimulus until the make and the break 
contractions are of the same height. Observe (1) that the induc- 
tion shock at the break of the current in the primary coil is a 252 
Laboratory Exercises in Physiology 
much stronger stimulus than that at the make; (2) that the 
height of the contraction increases with the strength of the stim- 
ulus up to a certain point (maximal contractions), but beyond 
that further increase in the strength of the stimulus does not 
intiuence muscular contraction. When the electro-magnet is 
shunted off from the primary circuit the make of the current 
in the primary may give a stronger induction shock than the 
break. 
With the secondary coil in a position to cause maximal con- 
tractions, close the short-circuiting key in the secondary, and 
make and break the primary current. Observe that the shocks, 
which are induced momentarily when the current is made or 
broken in the primary, are not felt by the nerve when the short- 
circuiting key is closed in the secondary. 
Open the short-circuiting key and place the secondary coil 
just beyond the position at which the minimal contractions were 
obtained with the weak break shocks. Make and break the prim- 
ary circuit* at intervals of a second or less. Observe that a sum- 
mation of subminimal stimuli may cause contraction although 
single stimuli are inadequate. 
(2) Galvanic or Constant Current: Connect the wires from 
the battery directly to the binding posts of the moist chamber 
with a simple key interposed for making and breaking the cur- 
rent. Make and break the current. Observe that contraction oc- 
curs at make and at break, but not during the flow of the con- 
stant current. Any sudden variation in the intensity of the 
current may act as a stimulus. 
If contraction occurs equally well both at make and break, .re- 
duce the strength of the current by using a single battery or by 
increasing the resistance to the flow of current. This may be 
done by causing the current from one dry cell to pass through 
the fine wire of the secondary coil. If the current obtained in 
that way is not weak enough to cause contraction at make and 
not at break, it may be still further reduced by connecting the 
wires from the battery to a rheocord with a key interposed and 
gradually increasing the strength of the current passing from 
the rheocord to the electrodes by moving the slider of the rheo- 
cord so that the resistance is increased. Observe that the make 254 
Laboratory Exercises in Physiology 
of a weak or moderately strong galvanic current is stronger than 
the break. 
177. Polarization of the Electrodes: Lead off copper elec- 
trodes from the short-circuiting key connected with the bat- 
teries. Lay the nerve on the electrodes and open the short- 
circuiting key. Allow the current to pass through the nerve for 
three to five minutes; then quickly detach the wires from the 
batteries; open and close the key repeatedly. Does contraction 
occur at the make and break of the circuit ? Stimulation in this 
case is due to the electrolytic change in the nerve (polarization). 
Platinum electrodes show much less polarization than those of 
copper or other metals, but when the constant current is passed 
through a nerve, non-polarizable electrodes of clay are used. 
178. Extensibility and Elasticity of Muscle: Fasten the 
femur of a muscle preparation securely in the large femur clamp 
and by means of the S-shaped hook fasten the tendon to the 
lever. Lower the after-loading screw so that the muscle can ex- 
tend. With the lever a little above the horizontal line bring it 
to write on the drum and turn the latter by hand for a distance 
of one-fourth of an inch. Now place a 20-gram weight in the 
pan under the lever and turn the drum the same distance as 
before. Remove the weight and again turn the drum as before. 
Is excised muscle perfectly elastic, i. e., does it return to its 
original length when the extending force is removed? 
Now place successively 20, 40, 60, 80, 100 and 120 grams in 
the weight pan, turning the drum by hand the same distance 
after each extension of the muscle. Does each increase of weight 
cause the same amount of extension of the muscle? 
Now remove the weights, taking 20 grams off at a time, and 
take a tracing after the removal of each weight to show the re- 
turn of the muscle. Does it return to the normal immediately 
after all the weights have been removed? Is excised muscle as 
perfectly elastic as a muscle in the living body? 
Repeat the experiment, using a spiral wire spring instead of a 
muscle, taking tracings as before. State the difference between 
th extensibility of muscle and that of a spring. 
179. The Simple Muscle Contraction or Twitch: Arrange 256 
Laboratory Exercises in Physiology 
a fresh nerve-muscle preparation in the moist chamber and 
place the nerve on the platinum electrodes. Arrange the inducto- 
rium for single induction shocks with the simple key in the pri- 
mary and the electro-magnet as a shunt to mark the point of 
stimulation. After-load the muscle lever with a 20-gram weight. 
Find a strength of current wljich gives a maximal contraction, 
both at make and at break, and bring the muscle lever and elec- 
tro-magnet to write exactly in the same vertical line. 
Set the drum at its fastest speed and let it make one turn. 
Cause the tuning fork to vibrate and bring it to write on the 
drum under the muscle lever. Each complete wave marks one 
one-hundredth of a second. While the drum is turning at its 
fastest speed stimulate several times with single induction shocks, 
taking care that the muscle relaxes after each contraction before 
the next stimulus is thrown in. What time is occupied by the 
entire simple muscle twitch and by each component part of it? 
Now take tracings in the same way, with the drum at the fast- 
est speed, of contractions caused by the make and break of the 
constant or galvanic current by detaching the wires to the moist 
chamber from the secondary coil and connecting them with the 
binding posts of the primary circuit from which the electro- 
magnet is shunted. Observe that the time of the simple muscle 
twitch is the same whether caused by the induced or the constant 
current. 
180. The Action of Curare and the Independent Irritability 
of Muscle: Pith a frog and destroy its brain. Expose the 
left sciatic nerve for a short distance in the thigh, taking great 
care that the nerve does not touch the skin and that the blood 
vessels about the nerves are not injured. Pass a ligature beneath 
the nerve and tie it tightly about the thigh near the knee so as 
to cut off the circulation in the leg below. Moisten the nerve 
and keep it from coming in contact with the skin. 
With a pipette inject into the dorsal lymph sac one or two 
drops of a 1 per cent solution of curare. After a few minutes 
the frog ceases to draw up the right foot when it is pinched or 
otherwise stimulated. (If necessary inject more curare, but be 
careful not to use any more than is necessary to cause motor 
paralysis.) 258 
Laboratory Exercises in Physiology 
Arrange the induction coil for single shocks with the hand 
electrodes attached to the secondary coil. Expose both sciatic 
nerves up to the pelvis, taking care that the nerves do not touch 
the skin. Use a strength of current that can be felt by the 
tongue. Stimulate the right sciatic nerve and observe that it 
does not cause contraction if enough curare has been given to 
cause complete paralysis. 
Now remove the skin from both legs and stimulate the gas- 
trocnemius muscle of the right leg directly by applying the 
hand electrodes. Does contraction result? 
Now apply the electrodes to the left sciatic nerve above the 
level of the ligature which was tied around the left thigh. Ob- 
serve that this nerve trunk has not been paralyzed, although 
the curare has reached it through the blood and lymph. If cur- 
are does not act upon the nerve or muscle fibers, how does it 
cause paralysis? 
Does the muscle possess irritability independently of the nerve 
fibers and motor nerve plates ? 
181. Comparative Irritability of Muscle and Nerve: Using 
the same curarized frog, place the electrodes beneath the left 
sciatic nerve so as to stimulate the left gastrocnemius indirectly, 
i. e., through its nerve. Find the minimal stimulus which will 
cause distinct contraction. Now stimulate the right gastroc- 
nemius with the same strength of stimulus, applying the hand 
electrodes directly. Does it contract? Increase the strength of 
the stimulus gradually until contraction occurs. Observe that 
the irritability of the muscle is considerably less than that of 
the nerve, even when the latter is reduced below the normal by 
anemia. 
Now apply the electrodes directly to the left gastrocnemius 
and find the minimal strength of stimulus which will cause con- 
traction. Apply the same strength of stimulus to the right 
gastrocnemius and observe that it is not felt because the motor 
nerve plates have been paralyzed. Increase the strength of the 
stimulus gradually until contraction occurs. Ordinarily when 
an electrical stimulus is applied directly to a normal muscle, 
is it the muscle or the nerve plates that are stimulated? 260 
Laboratory Exercises in Physiology 
182. Influence of Veratria Upon Muscle Contraction: Pith 
a frog and destroy its brain. Into the dorsal lymph sac in- 
ject four or five drops of a 1.0 per cent solution of veratria sul- 
phate. Wait a few minutes until the characteristic effect upon 
the muscle shows itself by the prolonged contraction and slow 
relaxation which follows when the toes are pinched. 
Now take a nerve-muscle preparation and after-load the mus- 
cle as usual. Arrange the induction coil for single induction 
shocks with the electro-magnet as a shunt in the primary cir- 
suit. Adjust the secondary coil to give maximal break contrac- 
tions. Bring the electro-magnet to write on a vertical line with 
the muscle lever. Set the drum at the fastest speed and let it 
make a complete turn; stimulate with a single induction shock 
and wait until the muscle relaxes completely before stimulating 
a second time. While the drum is going at the same speed take 
a time tracing in tenths of a second or in seconds. Observe that 
the latent period and height of contraction are unchanged, but 
the period of relaxation is enormously prolonged. Observe that 
a second slower contraction follows shortly after the summit of 
the first shortening. Record the time in seconds occupied by the 
phase of relaxation and compare it with the normal. 
183. Influence of Load Upon Muscular Contraction: Make 
a fresh nerve-muscle preparation and place it in the moist 
chamber. Arrange the inductorium for single induction shocks 
by contact through the kymograph and a platinum foil. Adjust 
the secondary coil so that the break shocks cause maximal con- 
tractions and the make is not felt. With the drum at fastest 
speed take a tracing with the lever after-loaded only with the 
heavy weight pan, which weighs 20 grams. 
Now move the drum on the upright tube so that the tracings 
will fall at another point on the drum. Take another tracing 
with the weight pan alone. Mark the exact point of stimulation 
on the tracing by moving the drum slowly by hand so that a 
vertical line is made by the muscle lever at the point where the 
current in the primary circuit is broken. With the point of 
stimulation and the muscle lever in exactly the same position, 
take tracings successively with 1, 3, 5 and 6 20-gram weights in 
the weight pan, i. e., with loads of 40, 80, 120, 140 grams, re- 262 
Laboratory Exercises in Physiology 
spectively. Neither the point of contact in the primary circuit, 
nor the position of the tracings should be changed while the con- 
tractions are recorded. Let the muscle lever mark the base line 
beneath the tracings and, with the drum at the same speed as 
before, take a time tracing in hundredths of a second with the 
tuning fork under the other tracings. 
Before varnishing the tracing, mark on each curve the amount 
of weight lifted. Observe' (1) the effect of increasing the load 
upon the height of the contractions; (2) the change in the time 
occupied by the different parts of the myogram. 
184. Calculate the Work Done With Different Loads: 
After the tracing has been varnished and dried, measure in mil- 
limeters the height of each curve from the base-line. Multiply 
this by the total load lifted in each case, remembering that the 
weight pan weighs 20 grams. Divide the product by the mag- 
nification of the lever as the actual shortening of the muscle is 
much less than the height of the tracings. This is determined by 
dividing the length of the long arm of the lever (i. e., the dis- 
tance in millimeters from the fulcrum to the writing style) by 
the length of the short arm (i. e., the distance from the fulcrum 
to the hook to which the muscle is attached). The work done in 
gram-millimeters would be determined by the formula: 
. LoadXheight of tracing 
W ork= . 
Magnification of the lever. 
Observe that a moderate load improves the contraction, bnt 
that an excessive load rapidly diminishes the height of contrac- 
tion. Note that the greatest amount of work is not done with 
the greatest degree of shortening of the muscle. Make a protocol 
of the work done with each load: 264 
Laboratory Exercises in Physiology 
Height. 
Load. 
Work. 
20 grams. 
40 grams. 
80 grams. 
120 grams. 
140 grams. 
185. Influence of Temperature on Muscular Contraction: 
Isolate the gastrocnemius without the nerve. Fasten the femur, 
or the tissues about the knee, to the end of the rod in the lower 
part of the warming chamber. From the hook in the tendo- 
Achillis lead a fine copper wire up through the opening in the 
top of the muscle warmer and fasten it over the pulley on the 
muscle lever, so as to record the contractions. See that the wire 
is taut and that the muscle is after-loaded with a 20-gram weight. 
Fasten the glass tube of the muscle warmer in place. 
Arrange, the inductorium for single induction shocks with the 
electro-magnet as a shunt in the primary circuit to mark the 
point of stimulation. See that the muscle lever and the electro- 
magnet write on the same vertical line and take a tracing of the 
normal muscle contraction at the room temperature with the 
drum at fast speed. Record the temperature under the tracing. 
Now insert the thermometer into the muscle warmer so that 
the bulb is near the muscle, but does not touch it or the glass. 
Take a mixture of ice and salt in a beaker and surround the 
glass with it until the temperature of the air inside the chamber 
has been lowered 6° or 8° C. Take another tracing with the 
drum at the same speed as before and record the temperature 
under each tracing. As the temperature continues to fall, take 
a tracing for each reduction of 6° or 8° C until the temperature 
is reduced to 0°C. 
After the temperature has been lowered almost to the freezing 
point, gradually warm the glass back to the room temperature by 266 
Laboratory Exercises in Physiology 
immersing it in tap water at first; then heat a beaker cf water 
carefully until the temperature of the air in the muscle chamber 
finally reaches 40° C. Take tracings of the muscle twitch at 25°, 
30°, 35° and 40° C, with the drum at the same speed as before. 
Then gradually increase the temperature still farther until heat 
rigor begins. Observe that the muscle begin to shorten at 41° 
to 45° C and gradually continues to do so until a greater degree 
of shortening occurs than can be produced by the contraction of 
muscle. This is heat rigor and the properties of irritability and 
contractility are lost. The drum should be set at very low speed 
with low gear as soon as the heat rigor begins, so as to take a 
tracing of the shortening. 
The change in reaction of the muscle from alkaline to acid 
may be shown with the onset of heat rigor by injecting acid 
fuchsin into the lymph sac of the frog some time before its muscle 
is used for this experiment. This indicator is harmless and does 
not influence muscular contractions. When an acid reaction oc-> 
curs a distinct rose red color develops. 
Briefly describe the changes produced in the muscular con- 
traction by heat and cold, respectively, stating the temperature 
at which maximal contraction occurred and the temperature at 
which heat rigor began. 
186. The Myogram Taken With the Drum at Fast Speed: 
Arrange for single induction shocks by contact between a piece 
of platinum foil fastened to a spring on a separate stand, and 
a platinum wire tip attached to a brass rod which is held in a 
block beneath the drum. Fasten one wire from the batteries to 
.the binding post on the spring holding the plaintinum foil; con- 
nect the other wire from the batteries, through a simple key, with 
the base of the kymograph by means of the thumbscrew on one 
side. 
Adjust the brass rod in the block so that the platinum wire 
tip just touches the platinum foil as the drum revolves. Screw 
the drum up free from the friction disc by means of the screw 
at the top of the central post which holds the drum. 
Arrange a fresh nerve-muscle preparation in a moist chamber. 
Find a strength of current which causes maximal break shocks, 
without contraction at make. Bring the drum to the position 268 
Laboratory Exercises in Physiology 
where contact is macle between the platinum wire and foil; mark 
the point of stimulation by closing the key in the primary circuit 
and causing the muscle lever to write in a vertical line. Ar- 
range the tuning fork to mark hundredths of a second just under 
the muscle lever. 
One student should spin the drum by hand, and as it revolves, 
he should close the key in the primary circuit until one stimulus 
is applied. As soon as one contraction occurs, the key should be 
opened. At the same time, while the drum is revolving at the 
same speed, the other student should take a tracing of the tuning 
fork vibrations beneath the muscle contraction. As soon as the 
drum makes one complete turn, the tuning fork should be taken 
away so that the tracings do not overlap. 
Observe that the single muscle contraction only occupies 0.10 
second. After the tracing has been varnished drop vertical lines 
and measure the time occupied by (a) the latent period, (b) the 
period of contraction and (c) the relaxation. Record your re- 
sults. 
187. The Refractory Period in Voluntary Muscle: Adjust 
two rods with platinum tips in blocks under the drum so that 
they will strike the platinum foil within 0.01 of a second when 
the drum is turned at the same speed as in the last experiment. 
Raise or lower the drum so that the muscle lever will write on 
another part. With the key in the primary circuit open, turn 
the drum around once to make a base line. Find the strength of 
current which causes maximal break shocks and, turning the 
drum very slowly by hand, mark the two points of stimulation 
by vertical lines caused by maximal contractions. 
Spin the drum at fast speed as before. After one turn of the 
drum with the key closed, open the key in the primary and take a 
tracing of hundreths of a second with the tuning fork while the 
drum is moving at the same speed as before. Observe that when 
a second maximal stimulus is applied during the latent period 
there is no change in the height or duration of the contraction, i. 
e., the second stimulus is not felt. There may be summation of 
two submaximal contractions falling within the latent period. 
188. Summation and Superposition of Contractions: (1) 
With the apparatus arranged as in the last experiment, separate 270 
Laboratory Exercises in Physiology 
the platinum tips of the rods farther apart so that the second 
stimulus will fall during the period of contraction. Mark the 
points of stimulation by making contact with each of the wires as 
before, and take a tracing on another part of the drum while it 
is spinning at fast speed as in the previous experiments. Note 
that when the two stimuli fall during the period of contraction 
there is a summation, i. e., the two stimuli are "summed up" to 
cause a greater single contraction than can be produced by any 
single stimulus. 
(2) Separate the two points of stimulation still farther apart, 
so that the second stimulus falls early in the period of relaxation. 
Mark the two points of stimulation as before, and while the 
drum is spinning at fast speed, take a tracing on another part 
of the drum. 
Observe that the second contraction begins after the latent 
period which follows the second stimulus, from whatever point 
the lever happens to be when the stimulus is felt, i. e., the second 
contraction is superimposed on the first one. 
Observe that the refractory period of voluntary muscle is lim- 
ited to the latent period and that stimuli falling during the 
period of contraction are felt. How does this compare with 
heart muscle? (See experiment 89, page 138.) 
189. Tetanus: Arrange the inductorium for single induc- 
tion shocks; place the tetanometer and a simple key in the pri- 
mary circuit. The tetanometer can be made to vary the rate of 
stimulation by changing the length of the spring; also by chang- 
ing the strength of the induction shocks used, so that stimula- 
tion occurs both at make and break or only at the break. 
Find a strength of stimulus which causes maximal contrac- 
tions both at make and break. Set the clock work of the drum 
for fastest speed in each of the following tracings and by means 
of the short circuiting key in the secondary circuit only stimulate 
for a few seconds at a time so that the muscle does not become 
fatigued. Take short tracings in turn of the muscular contractions 
caused by 8,12. 16, 20, 24, 32, and 40 stimuli per second. (See that 
the strength of the stimulus used each time the rate of interrup- 
tion is changed, is sufficient to cause contractions, both at make 
and break, so that the number of stimuli will be double the num- 272 
Laboratory Exercises in Physiology 
bers on the tetanometer.) Record the rate of stimulation under 
each tracing. Take a time tracing in seconds with the drum at 
the same speed as before. 
Write a description of complete and incomplete tetanus, re- 
spectively, stating what rate of. stimplation is necessary to pro- 
duce the former. 
A muscle that is fatigued is more easily tetanized than a fresh 
muscle, because the time occupied by each twitch or contraction 
is prolonged by fatigue. A fresh muscle-nerve preparation stim- 
ulated successively by 16, 24, 32, and 40 stimuli per second shows 
less complete tetanus than a muscle which has been gradually 
fatigued. 
190. Fatigue of Muscle; the Change in the Myogram From 
Fatigue: Make a fresh nerve-muscle preparation and arrange 
it in the moist chamber with a 20-gram weight as an "after- 
load." 
Lead one wire from the batteries through a simple key to one 
of the posts of the induction coil for single induction shocks. 
Fasten the other wire from the batteries to the binding post con- 
nected with the platinum foil. Connect the base of the kymo- 
graph with the other binding post of the primary circuit for 
single induction shocks. Adjust the platinum foil so that each 
arm of the contact-maker at the top of the drum touches it in 
passing without catching. 
Find a strength of current which will cause maximal contrac- 
tions at the break, while the make shocks are not felt. When the 
drum is revolving at the fastest speed, the contact-maker will 
produce a stimulus every 1 Vi seconds. See that the clock work 
of the kymograph is wound up, and with the key in the primary 
open, let the drum revolve once to make a base-line. Now close 
the key in the primary and, turning the drum by hand, mark 
the point of stimulation by vertical tracings at each point of 
contact. 
Set the drum going at its fastest speed and let it make one turn 
before closing the key in the primary circuit. Without changing 
the position of any of the apparatus, close the key and let it 
continue until the muscle is completely fatigued and fails to 
contract when stimulated. Watch the muscle lever during the 274 
Laboratory Exercises in Physiology 
experiment, and if necessary, increase the strength of the stim- 
uli slightly by shoving up the secondary coil, or adjust the plati- 
num foil, so that a contraction results from each contact. After 
the muscle is completely fatigued take a time tracing in tenths 
of a second with the drum at fastest speed. 
Observe the changes in the character of the contractions as 
fatigue develops: (a) the diminution in height; (b) the dispro- 
portionate prolongation of the relaxation period; (c) the slug- 
gish contraction, and (d) the prolonged latent period. 
191. The Effect of Load on Fatigue: (1) Make a fresh 
nerve-muscle preparation and after-load with a 20-gram weight. 
Disconnect the wires from the base of the kymograph and plati- 
num foil and lead wires from the time-marking binding posts on 
the table through two dry cells and a simple key to the binding 
posts of the inductorium for single induction shocks. These will 
be made at intervals of one second by the large clock. 
Set the drum for very slow speed by loosening the thumb 
screw at the base of the kymograph and shifting the brass rod 
to change the gear. Now set the drum at such speed that 'the 
contractions make separate and distinct vertical lines with very 
little space between them. Only the height of the contractions 
and the time necessary to produce complete fatigue are recorded 
in this experiment. 
Find a strength of current which gives maximal contractions 
at break but none at make. See that the clock work of the kymo- 
graph is wound up and let the drum revolve slowly until com- 
plete fatigue results from the frequent stimuli, i. e., until the 
muscle no longer shortens on stimulation. 
If the muscle is allowed to rest for a few minutes before 
starting the tracing, the first few contractions increase in height, 
like a staircase. This is due to the improvement from exercise. 
Somewhere in the series of contraction the condition of con- 
tracture or maintained contraction which is so characteristic of 
fatigue may be seen by the incomplete relaxation and failure of 
the lever to return to the base line. This may occur early or 
late in the development of fatigue, the muscles of different in- 
dividuals varying greatly. 
(2) Make a fresh nerve-muscle preparation from the other 276 
Laboratory Exercises in Physiology 
leg of the same frog whose muscle was used in the last experi- 
ment ; after-load with 60 grams, instead of 20 grams. Find a 
strength of stimulus which causes maximal contractions at break 
but none at make. Set the drum at the same speed as in the last 
experiment and take a tracing of the contractions produced at 
the same intervals of time. How does an increase in the load 
influence the time necessary to produce complete fatigue? 
192. The Seat of Fatigue: Make a fresh nerve-muscle prep- 
aration. Arrange the induction coil for a tetanizing current 
with a simple key in the primary circuit. Connect the binding 
posts of the secondary coil with the central binding posts of the 
pole-changer with its cross wires removed. From one side of the 
pole-changer lead wires to the electrodes in the moist chamber 
for stimulating the nerve; from the other side lead one wire to 
the metal holding the muscle in the moist chamber, and the 
other to the muscle lever for sending an induced current 
through the muscle directly. 
Find a strength of current which causes maximal contractions 
when applied to the nerve and also when applied directly to the 
muscle. Stimulate through the nerve until the muscle ceases to 
respond. Then reverse the pole-changer and stimulate the mus- 
cle directly. Observe that the contraction occurs, showing that 
the muscle is not as easily fatigued as the nervous structures 
which control it. Experimentally it has been shown that the 
motor nerve plates are the first to show fatigue in a nerve-muscle 
preparation, while the nerve fibers resist the fatigue much longer 
than the muscle. See experiment 204, p. 300, on fatigue of nerve. 
193. Fatigue in the Living Human Body From Voluntary 
Contractions: Arrange the ergograph so that the lever writes 
upon the drum. Set the drum at very slow speed by changing 
the gear. Place the hand on the block and with the tapes tie 
all the fingers securely except the index finger. Fasten the 
point of the upright rod in the middle hole of the spring and 
see that the finger-rest is screwed down against the index finger 
when it is at rest. 
Maximal voluntary contractions of the abductor idicis should 
be made every second until fatigue is complete. The time can 278 
Laboratory Exercises in Physiology 
be indicated by beats of the metronome or by the magnet con- 
nected with the time-marking current. When fatigue is com- 
plete, the belly of the abductor indicis should be stimulated di- 
rectly by means of single induction shocks passed through it 
from brass electrodes covered with cotton wet with salt solu- 
tion, which should be placed on opposite sides of the muscle. 
Observe that contraction occurs from direct stimulation after 
voluntary effort fails. Fatigue in the living body first shows 
itself in the spinal neurones. 
194. Nature of Voluntary Muscular Contraction: Change 
the position of the drum so that the ergograph writes upon a 
clear space and set the latter for fastest speed. Make several 
maximal voluntary contractions of the abductor indicis. Some 
of these should be made in the shortest time possible; others 
should be prolonged for several seconds. Connect the electro- 
magnet with the time current, with the drum at the same 
speed as before and record seconds under the tracing. Observe 
(1) the time occupied by the quickest ordinary voluntary con- 
traction and compare it with the time of the single muscle twitch 
produced by an induction shock; (2) that the tracings of the pro- 
longed contractions show small waves like an incomplete tetanus, 
showing that voluntary contractions are made up of separate 
contractions fused together. 
195. Current of Injury or Demarcation Current: Destroy 
the brain and spinal cord of a frog and remove the skin from 
the hind legs. Dissect out the sciatic nerve without injuring the 
thigh muscles and cut it as high in the pelvis as possible. (Use a 
pointed glass rod with a smooth tip instead of a metal seeker and 
do not let metals touch the nerve or muscle except in cutting 
them.) 
Before cutting the thigh muscles, lift the free end of the sci- 
atic nerve and let it fall longitudinally upon the uninjured sur- 
face of the triceps muscle. Observe that no contraction of the 
gastrocnemius follows, because there is no current in the thigh 
muscles to stimulate the nerve. 
Now cut the thigh muscles transversely a little above the mid- 
dle of the thigh. Lift the nerve with the glass rod so that its 280 
Laboratory Exercises in Physiology 
trunk rests upon the uninjured surface of the muscle and the 
free end of the nerve falls upon the surface of the transverse 
section through the muscles. Observe that the contraction of 
the leg muscles occurs each time the nerve touches these two 
points of the injured muscle, i. e., the make or closing of the de- 
marcation current stimulates the nerve. Sometimes, when the 
nerve is very irritable and there is a strong current of injury, 
contraction also occurs when the current is broken by lifting it 
from the injured surface. Usually the result is like the stimula- 
tion by a weak constant or galvanic current from any other 
source, i. e., contraction occurs at make but not at break. Re- 
peat the observation a number of times and record the results. 
196. Current of Action: Make two nerve-muscle prepara- 
tions leaving the foot and leg attached to each. Only handle the 
nerves with the glass rod or camel hair brush. Place them on a 
glass plate. Let the nerve of one (B) rest upon the gastroc- 
nemius of the other (A). Find the minimal induction shocks 
which produce maximal contractions when the hand electrodes 
are applied to the nerve of A. Be careful not to use a greater 
strength of current than necessary in order that there may be 
no escape of current from the electrodes. Observe that the 
muscle B contracts each time the nerve of A is stimulated. 
The nerve of the preparation B is stimulated by the current of 
action in the muscle of preparation A. 
Now use the tetanizing current for stimulating the nerve of 
A. Observe the secondary tetanus in the muscle B produced by 
the series of currents of action in the muscle A. 
Pinch the free end of the nerve A with forceps. Observe that 
each contraction of the muscle A produced by the mechanical 
stimulation of its nerve, causes a secondary contraction in B 
from the current of action in A. 
To prove that the nerve of B does not receive electrical stim- 
ulation from the electrodes connected with the induction coil, tie 
a ligature, moistened with physiological salt solution, about the 
nerve of A near to the muscle and stimulate the nerve of A with 
a tetanizing current as before. The ligature crushes the nerve 
fibers and blocks the transmission of nerve impulses, but does 
not prevent the conduction of an electrical current, i. e., it 282 
Laboratory Exercises in Physiology 
severs the continuity of the nerve physiologically, but net phy- 
sically. 
197. The Capillary Electrometer: The current of injury in 
the voluntary muscle of the frog and the current of action in 
the frog's heart will also be demonstrated by the capillary elec- 
trometer which is used to detect feeble electrical currents. In 
the one case one non-polarizable electrode is placed on the in- 
jured surface and the other on the uninjured surface; in the 
other case one electrode is placed on the base and the other on 
the apex of the heart. 
One electrode is then connected with the mercury and the 
other with the dilute sulphuric acid of the electrometer with 
a short-circuiting key interposed. Note the change in the 
level of the meniscus of mercury in the capillary tube, as ob- 
served under the low power of the microscope, when the short- 
circuiting key is opened. It always changes from a higher to 
a lower potential, the direction being the reverse of what it 
appears to be under the microscope. As soon as the key is 
closed the meniscus returns to the normal. 
PHYSIOLOGY OF NERVE. 
198. Relative Excitability of Nerves to Flexors and Ezten- 
sors: Pith a frog and with stout scissors cut across the spinal 
column about its middle. Cut the abdominal walls parallel to 
the pelvis and remove the viscera. Strip the skin from the 
legs. Lay the preparation on its dorsal surface and place one 
sciatic plexus on the shielded electrodes. 
Arrange the inductorium for a tetanizing current. Starting 
with the secondary coil at right angles to the primary, grad- 
ually increase the strength of the current until feeble con- 
tractions occur. Observe that the flexor muscles are the first 
to respond to the weak stimuli. 
Now increase the strength of the current considerably and 
observe that the extensors are more irritable to strong stimuli 
than the flexors. 
199. Distribution of the Nerve Fibers of the Sciatic Plexus' 
Separate the three roots of the sciatic plexus so that each 284 
Laboratory Exercises in Physiology 
one in turn can be stimulated separately, beginning with the 
upper (outer) root. Use the shielded electrodes and start with 
induction shocks which are too weak to be felt. Gradually 
increase the strength of the current until contractions occur. 
Observe that the upper (outer) root supplies the muscles mov- 
ing the thigh at the hip joint; the middle root causes motion 
of the leg upon the thigh at the knee joint; the lower (inner) 
root controls the lower muscles, causing movements at the 
ankle joint. 
200. Effect of CO2 Upon Nerve, Showing That Irritability 
and Conductivity are Separate Properties: Arrange the induc- 
torium for a tetanizing current and connect the secondary 
coil with the central binding posts of the pole-changer without 
cross wires. From one side of the pole-changer lead off hand 
electrodes; from the other side lead wires to the needle elec- 
trodes in the cork of the gas chamber. 
Connect the longer glass tube of the gas chamber with the 
short glass tube of the wash bottle connected with the gas 
generator by means of small rubber tubing. Place the gas 
chamber on a filter paper moistened with physiological salt 
solution on the cork plate and hold it in position by means 
of pins pressed into the cork plate. 
Now dissect out the sciatic nerve up to the vertebral column 
in a good-sized frog and cut it as high in the pelvis as possible. 
Free the nerve down to the knee; then cut across the thigh 
muscles and divide the femur at its middle, leaving the gas- 
trocnemius intact with the foot and leg. Tie a silk thread to 
the free end of the nerve, and by means of this, pass the nerve 
through the holes in the gas chamber, until the knee is close 
to the chamber. See that the nerve touches the electrodes 
within the gas chamber. Close the holes in the gas chamber 
by kaolin moistened with physiological salt solution without 
injuring the nerve. Fasten the femur in the femur clamp 
on the frog plate so that the foot is flexed on the leg by its own 
weight. 
Find a strength of current that is just sufficient to cause 
tetanus (a) when the hand electrodes are applied to the nerve 
above the chamber, and (b) when the nerve is stimulated by 
the electrodes within the chamber. 286 
Laboratory Exercises in Physiology 
Now pour some 10 per cent HC1 into the CO, generator 
through the thistle tube. After the CO, has passed through 
the gas chamber for several minutes, stimulate at both points 
as before, using the same strength of current. Observe that 
the stimulation by the electrodes within the chamber fails to 
cause contraction, or only does so with a very strong current, 
while practically the same strength of current that was first 
used causes tetanus when applied to the nerve above the gas 
chamber. This shows that CO2 lessens or destroys the irri- 
tability of the nerve fibers without changing the conductivity. 
Disconnect the CO, generator from the gas chamber and 
stimulate the nerve with the electrodes in the gas chamber again. 
Observe that the irritability soon returns to the normal after 
the CO, escapes. 
Connect an Erlenmeyer flask containing a small amount of 
alcohol with the gas chamber. Vaporize the alcohol by placing 
the flash in a pan of hot water. After several minutes stimulate 
at both points as before, increasing the strength of current if 
necessary. Alcohol diminishes the irritability somewhat, but 
almost entirely destroys the conductivity. 
Disconnect the rubber tubing from the gas chamber so that 
the vapor of the alcohol may escape. Again stimulate the 
nerve at both points as before, and observe that the stimulation 
above the chamber is again felt, so that the result obtained 
with alcohol is due to diminished conductivity within the cham- 
ber and not to diminished irritability near the free end of the 
nerve. 
201. Electrotonus; Effect of the Constant or Galvanic Cur- 
rent Upon the Vital Properties of Nerve: 
A. ALTERATIONS IN EXCITABILITY. 
From the binding posts of the time-marking current, lead 
wires through two dry cells to the binding posts of the primary 
coil of the inductorium for single induction shocks with a sim- 
ple key interposed and the electro-magnet arranged as a shunt. 
Connect the secondary coil with the platinum electrodes in the 
moist chamber. These electrodes are to be used as the stim- 288 
Laboratory Exercises in Physiology 
ulating electrodes to determine changes in the excitability, 
and should be placed next to the muscle. 
From two other dry cells lead wires through a simple key 
to the central binding posts of Pohl's commutator or pole- 
changer with the cross wires in. From the two binding posts 
on one side of the pole-chamber lead wires to the non-polar- 
izable electrodes in the moist chamber. These are the elec- 
trodes for producing the changes of electrotonus during the 
passage of the constant or galvanic current, and must be ap- 
plied to that part of the nerve farthest away from the muscle. 
See that the set screws in the blocks for holding the boot 
electrodes are placed toward the foot of the boot and not away 
from it. The boots must be tilted at an angle of 45 degrees 
with the floor of the moist chamber, so that the heel of the 
boot is the most dependent part and the toe is level with the 
top of the boot. This prevents the ZnSO4 solution from reach- 
ing the toe of the boot. The cup on the foot of the boot must 
be filled with physiological salt solution and the nerve should 
rest on the very tips of the toes of the boot electrodes. 
Determine the position of the reverser which gives an as- 
cending current, i. e., with the anode nearest the muscle, and 
mark that side of the commutator "a" with chalk; mark the 
other side "d" for a descending current, i. e., when the cathode 
is nearest the muscle. 
Make a nerve-muscle preparation with as long a stretch of 
nerve as possible and place, it in the moist chamber with the 
nerve resting on both pairs of electrodes, the stimulating elec- 
trodes from the induction coil being nearest the muscle. Place 
some moist filter paper in the moist chamber to prevent drying 
of the nerve. 
Bring the muscle lever to write upon the drum with the 
electro-magnet on the same vertical line beneath it. 
1. Cath electrotonus or Increased Excitability at the Cathode: 
(a) Close the key in the primary circuit of the induction 
coil and find the position of the secondary coil at which min- 
imal break contractions occur as the current is interrupted 
every second, or every other second, by the clock. Set the 
drum going at slow speed and take a tracing of several minimal 
or submaximal contractions until they are uniform in height. 290 
Laboratory Exercises in Physiology 
Arrange the reverser of the pole-changer for a descending 
current. Now close the key of the galvanic, or as it is here 
called, the polarizing current, for ten to twenty seconds. Ob- 
serve that the contractions caused by the induced or stimulating 
currents are greatly augmented and may be maximal, as a con- 
dition of cathelectrotonus is produced in the nerve near the 
stimulating electrodes. 
(b) Now lessen the strength of the induced currents by 
changing the position of the secondary coil until contractions 
just fail to occur (subminimal stimuli). Set the drum at the 
same speed as before, with the electro-magnet marking the 
points of stimulation. Again close the descending galvanic or 
polarizing current. Observe that the subminimal stimuli now 
produce contractions, because the irritability is increased in 
the neighborhood of the cathode, i. e., the nerve near the stim- 
ulating electrodes is in a state of cathelectrotonus. 
Note.-The galvanic or polarizing current should not be too strong, 
nor should it be passed through the nerve too long at a time, or the 
irritability may be lessened after a brief period during which it is 
increased. Even with a current of moderate strength the inceased 
irritability at the cathode is of short duration and is often quickly 
followed by the opposite condition. As an after-effect the irritability 
is decreased after the passage of the polarizing current. 
The alteration in the excitability at the cathode is much less con- 
stant than that which takes place about the anode. 
2. Anelectrotonus or Decreased Excitability at the Anode: 
(a) Increase the strength of the stimulating induction shocks 
by changing the position of the secondary coil, until the weak- 
est stimulus is found which will cause maximal contractions. 
Set the drum going at slow speed as before, and take a tracing 
to see that the contractions are uniform in height. 
Reverse the pole-changer, so that the anode is next to the 
stimulating electrodes, and close the key, causing an ascending 
galvanic or polarizing current to pass through the nerve. Ob- 
serve that the induced or stimulating currents, if not too strong, 
either fail to cause contractions or only produce submaximal 
contractions as the irritability is lessened in the neighborhood 
of the anode. 
(b) Find the position of the secondary coil which will give 
stimulating currents of such strength as to cause minimal or 292 
Laboratory Exercises in Physiology 
submaximal contractions. Set the drum going at slow speed 
as before and take a short tracing to get contractions of uni- 
form height. Without stopping the drum, close the key in the 
ascending galvanic or polarizing current. Observe that the 
contractions cease. The points of stimulation are indicated by 
the electro-magnet. Open the key in the polarizing current 
while the drum is still going, and observe that the condition 
of anelectrotonus, or decreased excitability, is followed by a 
condition of increased irritability as an after-effect after the 
passage of the galvanic current. 
B. CHANGES IN CONDUCTIVITY. 
1. With all the connections from the batteries remaining 
the same as in the last experiment, place the platinum or stim- 
ulating electrodes from the induction coil near the free or 
central end of the nerve; bring the two non-polarizable elec- 
trodes connected with the galvanic current as near the muscle 
as possible, i. e., between the stimulating electrodes and the 
muscle. 
Find the position of the secondary coil which will give in- 
duction shocks of such strength as to cause submaximal or 
maximal contractions as the current is interrupted in the pri- 
mary circuit by the time marker. Set the drum at slow speed 
and take a short tracing. 
(a) Without stopping the drum, close the key in the as- 
cending galvanic or polarizing current. Observe that the con- 
tractions cease. 
(b) Now open the galvanic current and change the reverser 
of the pole-changer so as to give a descending current. Again 
close the key in the galvanic current and observe that the con- 
tractions cease. 
The conductivity of the nerve is lowered by the galvanic cur- 
rent between the muscle and the stimulating electrodes, thus 
blocking the nerve impulses from the latter. As the conduc- 
tivity is lowered so as to produce a complete block when the 
galvanic current is passed in either direction, the failure to get 
contractions can not be due to the changes in irritability. 
2. Now place the stimulating electrodes midway between 294 
Laboratory Exercises in Physiology 
the non-polarizable electrodes and place these as far apart as 
the length of the nerve will permit. 
Find the position of the secondary coil which will cause 
submaximal or maximal contractions. Set the drum at slow 
speed and take a short tracing. 
(a) Without stopping the drum, close the key of the de- 
scending constant or polarizing current, i. e., produce a condi- 
tion of cathelectrotonus between the muscle and the stimulating 
electrodes which are placed at the nodal point. Observe that 
the contractions cease. 
(b) Open the galvanic current and change the reverser of 
the pole-changer so that the anode will fall between the muscle 
and the stimulating electrodes. While the drum is still going, 
close the key of the ascending constant current and observe that 
the contractions cease; 
This shows that the conductivity is decreased, both at the 
anode and at the cathode, the degree depending upon the 
strength of the galvanic or polarizing current. The decreased 
conductivity at the anode may come on more gradually than 
at the cathode. 
C. ELECTROTONIC CURRENT. PARADOXICAL CONTRACTION. 
Pith a frog and expose its sciatic nerve. Sever the nerve as 
high up in the pelvis as possible and trace it down to the knee. 
Observe the two branches into which it divides. Determine 
which branch goes to the gastrocnemius and divide the other 
one as near to the knee as possible. 
Stimulate the central end of the divided branch by a gal- 
vanic current and observe that the muscle supplied by the other 
division of the nerve contracts. This is due to the electrotonic 
current set up in the nerve by the galvanic or polarizing cur- 
rent. Contractions can not be produced by stimulating the 
divided branch of the nerve by a mechanical stimulus or by 
induction shocks. The stimulation of efferent fibers can not, 
therefore, be due to the current of action. This electrotonic 
current is a physical change, which may be detected by the 
galvanometer in a dead nerve, while the alterations of the ir- 
ritability and conductivity produced by the galvanic current 
are changes of the physiological or vital properties of the nerve. 296 
Laboratory Exercises in Physiology 
202. Pfliiger's Contraction Laws or the Stimulating Effect 
of the Galvanic Current: Make a'fresh nerve-muscle prepara- 
tion and place it in the moist chamber with the nerve on the 
non-polarizable electrodes. Tilt the boot electrodes as in the 
preceding experiment so that the toe is level with the top of 
the boot. The nerve should rest on the tip of the toe of each 
electrode. See that there is plenty of moist filter paper in 
the moist chamber to prevent drying. 
Connect the wires from one or two batteries with the central 
binding posts of the pole-changer with a simple key interposed. 
From the ginding posts on one side of the pole-changer lead 
wires to the rheocord. From the rheocord, as a shunt, lead 
two wires to the non-polarizable electrodes in the moist cham- 
ber. Arrange the pole-changer for a descending current. 
1. Make and break the current, gradually increasing the 
resistance to the current that passes through the rheocord, until 
contractions occur at make but not at break. 
Now reverse the direction of the current and stimulate as 
before, gradually increasing the strength of the current shunted 
off to the nerve by increasing the resistance to the current pass- 
ing through the rheocord, until the contraction again occurs 
at make but not at break. This is the response obtained by a 
weak current and shows that the increase in excitability caused 
by the development of cathelectrotonus at the make is greater 
than that caused by the disappearance of the anelectrotonus 
at the break of the galvanic current. 
2. Now increase the strength of the current shunted off to 
the nerve, by increasing the resistance offered to the current 
from the batteries through a greater length of German silver 
wire in the rheocord, until the contractions occur both at the 
make and at the break of the ascending and of the descending 
current. This is the reaction obtained by a current of 
medium strength. 
3. The strength of current should be still further increased. 
If this can not be done by increasing the resistance of the 
rheocord still further so as to shunt off more of the current 
from the batteries to the nerve, the battery current may be 298 
Laboratory Exercises in Physiology 
connected directly to the non-polarizable electrodes with the 
pole-changer and a simple key interposed. Observe that a 
strong descending current gives contractions at make, but not 
at break, while a strong ascending current gives contractions at 
break, but not at make. > 
The contractions obtained with the galvanic current vary 
with the electrotonic changes produced and depend upon the 
following conditions: (1) the intensity of the current; (2) 
the direction of the current; (3) the make or break of the cur- 
rent. Tabulate the results obtained below: 
Ascending. 
Descending. 
Strength of Current. 
Make. 
Break. 
Make. 
Break. 
Weak. 
Medium 
Strong. 
203. Reactions With the Galvanic Current in the Living 
Body: The galvanic current is used practically by placing a 
large flat indifferent electrode anywhere on the body, and then 
a small electrode placed over a muscle or nerve is used as a 
stimulating electrode. The latter is connected, in turn, with 
the cathode and the anode. Both electrodes must be covered 
with cotton wet with strong salt solution so as to lessen the 
resistance offered by the skin. The result obtained depend 
upon the electrotonic changes produced at the physiological 
anodes and the physiological cathodes in each case and the 
density of the current at the stimulating electrode. 
This may be demonstrated in man by using from eight to ten 
or more dry cells arranged in series. The commutator or pole- 
changer is used as a convenient means of changing the direction 
of the current. The stimulating electrode is placed over the 
ulnar nerve between the internal condyle and olecranon. 
State the order in which the following occur in the normal 
body: anodal closing, anodal opening, cathodal closing, cath- 
odal opening. 300 
Laboratory Exercises in Physiology 
204. Fatigue of Nerve: Arrange a nerve-muscle prepara- 
tion in the moist chamber. Place the non-polarizable electrodes 
between the stimulating (platinum) electrodes and the muscles. 
Stretch the nerve across both pairs of electrodes. 
Make another nerve-muscle preparation, but do not isolate 
the gastrocnemius. Expose the sciatic nerve down to the knee 
and sever it as high as possible; cut the femur at is middle, 
but leave the foot and leg attached. Hold the femur so that 
the nerve of the second preparation rests upon the stimulating 
electrodes with the free end of the other nerve. 
With the ends of both nerves on the stimulating electrodes, 
find the strength of induction currents necessary to produce 
tetanus in both preparations. Now make the galvanic current 
so that the impulses produced by the tetanizing current will not 
be able to reach the muscle of the first preparation on account 
of the "block" in conductivity caused by the galvanic current. 
Stimulate both nerves with a tetanizing current until the 
muscle of the second preparation ceases to contract and the 
toes do not move; then break the galvanic current. Observe 
that contraction occurs in the first preparation. 
Both nerves were stimulated by the same tetanizing current 
until the second preparation showed fatigue. The nerve im- 
pulses could not reach the muscle of the first preparation on 
account of the block produced by the diminished conductivity 
during the passage of the galvanic current. This shows that 
the nerve fibers are not readily fatigued, in fact they are not 
fatigued as easily as muscle. 
In a previous experiment on muscle (experiment 192) it was 
shown that the muscle responds to direct stimulation after it 
fails to respond to indirect stimulation through its nerve. If 
the first seat of exhaustion is not in the muscle or nerve, it 
must be in the motor nerve plates. 
205. Reactions of Degeneration: The electrical reactions 
of muscles after degeneration of their nerves will be demon- 
strated in two dogs. The sciatic nerve of one is cut high up 
and a small segment is excised to prevent rapid regeneration. 
The sciatic nerve of the other is merely severed and the ends 
are allowed to fall in close apposition in the wound so that re- 302 
Laboratory Exercises in Physiology 
generation can take place without any difficulty. The electrical 
response of the paralyzed gastrocnemius as compared with the 
normal side is studied at intervals of two to four weeks after 
the operation. 
Observe and record (1) the relative excitability of the par- 
alyzed and unparalyzed muscle to electrical stimulation; (2) 
the irritability to the induced (faradic) current as compared 
with the constant (galvanic) current; (3) the "qualitative" 
change with the galvanic current, i. e., the anodal closing con- 
traction (An. C. C.) as compared with the cathodal closing 
(C. C. C.). Write an account of the reaction of degeneration 
as observed from time to time in each one and explain the dif- 
ference between them 
206. Structural Changes During Degeneration and Regen- 
eration of Nerve Fibers: Study carefully the longitudinal sec- 
tions of the peripheral end of the sciatic nerve after section 
of the nerve. 
(a) Compare the appearance of the peripheral end in the 
early stage of degeneration (one week after the section) with 
the central (normal) part of the nerve. Both sections have 
been stained in exactly the same way by Pal's method. 
(b) Observe the appearance of another nerve showing com- 
plete degeneration four weeks after section. The electrical re- 
actions of degeneration were complete in this case. 
(c) Study the appearance of a sciatic nerve showing re- 
generation, twelve weeks after section and primary union. 
Observe that the fibers of the peripheral end do not differ from 
the normal fibers of the central end. Note the thickening 
and cicatricial tissue at the point of section; that many delicate 
axis cylinder processes grow out from the central end of this 
scar tissue; many of these can be traced into the peripheral 
end of the nerve; some have become curled and twisted by the 
scar tissue so that they do not reach the peripheral end of the 
nerve trunk. The reactions of degeneration had been complete 
three weeks after section of the nerve in this experiment. At the 
time the animal was killed (twelve weeks after the operation) 
the electrical reactions were normal and the motor paralysis 
had completely disappeared. 304 
Laboratory Exercises in Physiology 
Make drawings and describe the appearance of each of these 
sections. 
REFLEXES. 
207. Preparation and Observation of a Reflex Frog: Pith a 
frog with an ordinary pithing-pin, severing the medulla by 
moving the pin laterally, and destroy the brain by turning the 
pin forward into the cranial cavity. Be careful not to injure 
the spinal cord. The animal is in a condition of shock, i. e., 
there is a temporary depression or suppression of the spinal 
reflexes and nervous activities as the result of the sudden 
severe injury to the central nervous system. Physiological 
shock only lasts a short time and rapidly disappears, but 
clinically surgical shock is, as a rule, progressive and is charac- 
terized by a greater disturbance of the circulation. 
Observe the frog carefully in the condition of shock and com- 
pare with a normal frog placed under the bell-jar. Note (1) 
that the eyes are closed; (2) the posture is quite different; 
(3) the muscles are completely relaxed so that the extremities 
are not drawn up to the body; (4) the respiratory movements 
have ceased and there are no reflex movements even when the 
foot or toe is pinched. Record your observations. 
Observe the Reflex Frog: After waiting some time for the 
condition of shock to pass off, note (1) that the animal 
does not sprawl out as much as at first; (2) that the extremities 
are drawn up to the trunk; (3) reflex movements may be pro- 
duced by stimulating the skin or pinching a toe; (4) the animal 
does not sit as erectly as a normal frog and may not turn over 
on the ventral surface when placed on its back (compare this 
with a normal frog) ; (5) the eyes remain closed and there is 
an absence of respiratory movements; (6) note that when one 
leg is extended from the body it is promptly drawn back to its 
natural position, but some stimulus is necessary to cause move- 
ments. i. e., all activities are now reflex in nature. Spontaneous 
movements do not occur. Reflex actions may be carried on by 
the spinal cord alone without the brain. Write an account of 
your own observations on the reflex frog. 
208. Reflex Action: Pass an S-shaped hook through both Laboratory Exercises in Physiology 
306 
jaws and suspend the .reflex frog from a funnel ring. Pinch 
one toe feebly with the forceps, repeating the stimulation and 
gradually increasing the strength of the stimulus. Observe 
that a feeble stimulus causes slight movements of the part 
stimulated, and an increase in the intensity of the stimulus 
causes more extensive movements. Moderate stimulation only 
causes movements of the part stimulated, but a strong stimulus 
may cause movements of the other parts, the reflex activities 
extending (by irradiation) first to the opposite side of the cord 
and then to other segments of the spinal cord. When the stim- 
ulus is applied to the trunk near the median line the move- 
ments may be bilatral even from moderate stimulation. De- 
scribe the results of this experiment. 
209. The Purposive Character of Reflexes; Co-ordinated 
Reflexes: Moisten several small pieces of filter paper (not 
more than one-fourth of an inch square) with commercial 
acetic acid in a watch crystal. Have at hand a large beaker 
of water. With forceps pick up one of the pieces of paper, 
drain off any excess of the acid, and apply the paper to the 
skin on the inside of one thigh, taking care that it does not 
touch the opposite thigh. Carefully observe the' movements 
which result and then quickly wash the acid off by raising and 
lowering the beaker of water without handling the frog. 
Write an account of your observation. What part first 
showed reflex movements? Was it a simple reflex limited to 
the part stimulated, or was there irradiation to other spinal 
centers producing complex reflex movements? Were these move- 
ments co-ordinated? Was the reflex purposive or not? 
After a rest of five minutes, repeat this experiment, but hold 
the leg on the side to which the acid is applied. Observe the 
unsuccessfid efforts on the side stimulated, and the co-ordinated 
movements of the opposite side to remove the irritant. Wash 
the acid off as before and rest again. 
Now apply a piece of paper moistened with acetic acid to 
the skin of the trunk just above the anus. Observe the move- 
ments which are bilatral from the first, and which are co- 
ordinated in the beginning, but soon become inco-ordinate. 
Wash the acid off and record your observation. 308 
Laboratdry Exercises in Physiology 
210. The Effect of Strychnia Upon the Excitability of the 
Spinal Centers: With a fine pipette inject into the dorsal 
lymph sac of a reflex frog one or two drops of a 0.1 per cent 
solution of strychnia sulphate. Wait a few minutes for the 
drug to be absorbed. Now apply a light stimulus to some parts 
of the body (e. g., pinch a toe with the forceps). Observe that 
this is not followed by a simple reflex, but by complex, inco- 
ordinate movements of other parts. A feeble stimulus now 
causes irradiation throughout the spinal cord and may excite 
extensive convulsive movements or general spasms. This is 
due to an increase in the irritability of the spinal centers. 
Keep the frog quiet and free from any irritation. Observe 
that the spasms do not come on spontaneously, but from some 
slight stimulus which ordinarily would not excite reflex ac- 
tivity. Note that the extensor muscles are affected more than 
the flexors during the convulsive seizures. 
211. The Action of Chloral in Lessening the Excitability 
of the Spinal Cord: With a pipette inject five or six drops 
of a 1 per cent solution of chloral hydrate into the dorsal 
lymph sac of the frog used in the last experiment and wait for 
its absorption. 
From time to time stimulate the frog and if the exalted ex- 
citability of the spinal cord produced by the strychnia does not 
disappear, inject some more chloral until that result is ob- 
tained. 
Stimulate the frog in the same way as in the previous ex- 
periment and observe that the excitability of the spinal centers 
is diminished by chloral. 
212. The. Reflex Centers Contained in the Spinal Cord: 
Introduce the pithing-pin through the opening made between 
the skull and the first vertebra and pass it down the vertebral 
canal as far as it will go, destroying the entire spinal cord. 
After waiting some time for the shock to pass off, apply differ- 
ent stimuli and observe that it is impossible to obtain any 
reflex action because the "reflex arc" has been broken by de- 
struction of the reflex centers. 
Now remove the skin from the legs and expose the sciatic 
nerve. Stimulate the muscles and nerve by applying the hand 310 
Laboratory Exercises in Physiology 
electrodes from the induction coil and observe that both the 
nerve and the muscles are irritable. The failure to obtain re- 
flex action can not, therefore, be due to any loss of the vital 
properties of the nerves or muscles. 
213. Reflex Time as Determined by Tiirck's Method. Vari- 
ation in Reflex Activity: For this experiment use a frog that 
has been prepared two hours before the exercise so that there 
will be no delay in waiting for the shock to pass off. The 
cerebral lobes have been removed by section in front of the 
optic lobes, so that the latter bodies (representing the basal 
ganglia of the brain) remain intact with the spinal cord. 
Have at hand a beaker of 0.2 per cent sulphuric acid, a 
beaker containing 0.4 per cent sulphuric acid, and a large 
beaker of water. See that each one is correctly labeled. 
Connect the electro-magnet through two dry cells from the 
binding posts of the time current on the desk. This current 
will be interrupted 100 times per minute. 
Suspend the frog by an S-shaped hook from a support. With 
a glass rod gently hold one leg aside and immerse the other 
foot up to the ankle joint in 0.2 per cent sulphuric acid. Count 
the number of times the time-marking current is made by watch- 
ing the electro-magnet, between the application of the stimulus 
and the beginning of the reflex action in withdrawing the 
foot. Quickly wash the acid off by raising and lowering the 
beaker of water, taking care that the frog is not handled and 
does not touch the beaker. Note the reflex, time or spinal 
latent period in hundredths of a minute. After waiting three 
or four minutes, repeat the experiment and take the mean of 
the two observations as the average reflex time with a weak 
stimulus. Record your results. 
After another period of rest, repeat the experiment, using 
0.4 per cent sulphuric acid and washing the acid off each time. 
Make two observations and take the average. Observe that an 
increase in the intensity of the stimulus decreases the reflex 
time and may cause spinal irradiation. Record your observa- 
tions. 
214. Stimulation of the Reflex Inhibitory Centers of the 
Optic Lobes by Quinine: Inject into the dorsal lymph sac of 312 
Laboratory Exercises in Physiology 
the frog used in the last experiment a few drops of a 2 per cent 
solution of quinia bisulphate and wait several minutes for its 
absorption. 
Determine the reflex time by Turek's method, using the 
weaker acid and washing the acid off after each observation. 
Record the result, making two observations. 
Note that the reflex time has been prolonged considerably, 
showing that the reflex activity of the spinal centers has been 
depressed. 
Now destroy the medulla of the frog by introducing the pith- 
ing-pin between the skull and atlas and moving it laterally. This 
severs the connection between the optic lobes and the spinal 
centers. After waiting for the shock to pass off, again deter- 
mine the reflex time as before, taking the average of two observa- 
tions. Compare the reflex time now with the last observation 
and record the result. 
Observe that the reflex time has been lessened by removal of 
the optic lobes, showing that the decreased reflex activity under 
the influence of therapeutic doses of quinia is due to stimulation 
of the reflex inhibitory centers in the optic lobes (Setschenow's 
center). Overdoses of quinia may increase reflex activity, there- 
by lessening the reflex time, by directly stimulating the spinal 
centers. 
215. Reflex Time as Determined by Electrical Stimulation: 
Use the same frog as in the last experiment. Arrange the in- 
ductorium for single induction shocks with the simple key in 
the primary circuit. From the secondary coil lead off two very 
delicate wires and hook their free ends in the web of the right 
foot, taking care that the wires do not touch and thus short-cir- 
cuit the induced currents. Wait for the frog to become quiet 
after the handling and mechanical stimulation of the foot. 
Start with very weak single induction shocks which are not 
felt, making and breaking the current by the key in the primary, 
and gradually increase the strength of the induced currents until 
the stimuli are felt. Observe that the single induction shocks 
only cause a local twitching or contraction, but fail to cause re- 
flex movements. 
Now arrange the inductorium for a tetanizing current, using 314 
Laboratory Exercises in Physiology 
the same strength of current that caused local twitchings before. 
Close the key in the primary, and from the electro-magnet in the 
time-marking current, count the hundredths of a minute that in- 
tervene between the application of the stimulus and the begin- 
ning of the reflex movements. If necessary, increase the strength 
of the current until reflex action is produced. After a rest of a 
few minutes repeat the observation. Record the strength of the 
current used and the reflex time as indicated by the average of 
the two observations. Observe that the reflex time, as determined 
by electrical stimulation, is less than it is when dilute acid is 
applied to the skin. 
216. Number of Stimuli Required for Reflex Action: Let 
the secondary coil remain in the same position as in the last 
experiment with the wires attached to the frog's foot. Close the 
short-circuiting key in the secondary and arrange the inducto- 
rium for single induction shocks with the tetanometer in the 
primary circuit. Arrange the tetanometer, in turn, for 8, 16. 20 
and 24 stimuli per second and determine the reflex time with 
each rate of stimulation, allowing the frog to rest several minutes 
after each observation. The interruptions by the tetanometer 
may be started with the short-circuiting key of the secondary 
current closed; the reflex time is then determined by counting 
the hundredths of a minute, as indicated by the magnet in the 
time current, from the opening of the short-circuiting key in the 
secondary until the reflex movement starts. A determinate 
number of stimuli is necessary to elicit reflex action. Observe 
that the higher the rate of stimulation, the shorter the reflex 
time. Record your results. 
217. Inhibition of the Spinal Reflexes by Stimulating the 
Optic Lobes: For this experiment a frog is prepared for each 
table before the exercise as follows: the frog is etherized and 
the roof of the skull removed so as to expose the cerebral and 
optic lobes. The former are then removed. 
After the frog has recovered from the shock, determine the re- 
flex time by Turek's method, using 0.2 per cent sulphuric acid. 
After a period of rest, repeat the observation and take the aver- 
age of the two as reflex time with Setschenow's reflex inhibitory 
center'-in the optic lobes intact. 316 
Laboratory Exercises in Physiology 
Now apply one or two crystals of NaCI to each optic lobe, tak- 
ing care to remove any fluid or blood by sponging before the salt 
is applied so that it does not reach the medulla or spinal cord. 
Again determine the reflex time in the same way as before, and 
observe that it is greatly prolonged as the result of the inhibition 
of spinal reflexes caused by the chemical stimulation of Setseh- 
enow's center in the optic lobe. Record your observations. 
Now destroy the medulla by pithing between the skull and 
atlas, thus severing the connection between the optic lobes and 
the spinal cord. After the shock has passed off, again determine 
the reflex time, making two observations, and compare with the 
results obtained before. Record your results. 
218. Inhibition of Reflexes by Simultaneously Stimulating 
Different Parts: Suspend a reflex frog from a support and 
fasten the delicate wires from the secondary coil into the web 
of the right foot. Arrange the inductorium for a tetanizing cur- 
rent and find a strength of stimulus which will cause reflex 
movements of the right leg. 
Now determine the reflex time when 0.2 per cent sulphuric 
acid is applied to the left foot by Turek's method. Wash off the 
acid, wait a few minutes, and then repeat the observation, taking 
the average of the two. 
Now close the key in the primary circuit of the tetanizing cur- 
rent, and just as the short-circuiting key of the secondary cur- 
rent is opened to stimulate the right foot, again immerse the left 
foot in the 0.2 per cent sulphuric acid as before, and determine 
the reflex time. Repeat this observation. 
Observe that reflexes are inhibited, or the reflex time is pro- 
longed, when stimuli reach the spinal centers simultaneously 
from different parts of the body, i. e., when nerve cells are oc- 
cupied with one set of nerve impulses they are less sensitive to 
others. Record your results. 
TRACTS OF THE SPINAL CORD. 
219. Crossed. Pyramidal Tract: Study the section of a 
human spinal cord showing secondary degeneration of the 
crossed pyramidal tract, or according to the new nomenclature, Laboratory Exercises in Physiology 
318 
the fasciculus cerebrospinalis lateralis, at different levels of the 
cervical and thoracic cord. The specimens have been stained by 
a modification of Marchi's method. 
220. The Great Pyramidal Pathway in the Dog: The brain 
and spinal cord of a dog were taken out two weeks after the 
removal of the Rolandic or motor area of the cerebral cortex; 
the tissues were fixed in Muller's fluid and stained by Marchi's 
method. The motor fibers, which have undergone secondary de- 
generation, are stained black by the osmic acid while the normal 
undegenerated fibers do not stain. 
Sections have been made through the internal capsule, crus, 
pons, medulla and the cervical, thoracic and lumbar segments of 
the spinal cord. Study these sections carefully, and observe the 
position of the pyramidal fibers at each level; also the gradual 
diminution in the number of fibers as the crossed pyramidal 
tract extends down the cord. 
Note especially the position of these fibers in the upper part 
of the medulla, the decussation of fibers at the level of the decus- 
sation of the pyramids, and the position of the crossed pyramidal 
tract at the lower level of the medulla. 
221. Secondary Degeneration from Hemisection of the 
Spinal Cord: Lateral hemisection of the spinal cord of a cat 
has been made at the junction of the thoraic and lumbar seg- 
ments. Sections have been made at different levels above and 
below the lesion and stained by Marchi's method. 
Study these sections carefully. Observe the tracts which show 
ascending degeneration throughout the length of the cord, viz., 
(1) the fasciculus gracilis, formerly called the postero-median 
or column of Goll; (2) the fasciculus cerebellospinalis, or ac- 
cording to the old nomenclature, the direct cerebellar; and (3) 
the fasciculus anterolateralis superficial is, also known as the an- 
tero-lateral ascending or column of Gowers; also the tracts which 
only degenerate a short distance above the lesion, viz., the fas- 
ciculus cuneatus, formerly called the postero-external or column 
of Burdach, and the marginal zone of Lissauer. 
Note the extensive degeneration in the anterior and lateral 
ground bundles immediately above and below the lesion. 320 
Laboratory Exercises in Physiology 
Observe the descending degeneration in the fasciculus cere- 
brospinalis lateralis or crossed pyramidal tract below the lesion; 
also in the rubro-spinal tract. 
222. Secondary Degeneration After Section of the Posterior 
Column of the Spinal Cord: The posterior columns of the 
spinal cord of a eat have been cut, in one case between the last 
thoracic and first lumbar segments, and in another animal, be- 
tween the last lumbar and first sacred segments. The sections 
have been stained by Marchi's method. 
Observe the degeneration in the fasciculus cuneatus or column 
of Burdach for a short distance above the lesion. The degenera- 
tion in the fasciculus gracilis or column of Goll extends up to 
the medulla, although the number of degenerated fibers is much 
less in the upper segments. 
223. Secondary Degeneration in the Spinal Cord From Sec- 
tion of the Posterior Nerve Roots: The posterior nerve roots 
of the fifth and sixth lumbar nerves have been cut above the 
spinal ganglia on one side in a cat. The sections have been 
stained by Marchi's method. 
Study these sections carefully. Observe degeneration of the 
fasciculus cuneatus or column of Burdach and the column of 
Lissauer just above the section. The fasciculus gracilis or col- 
umn of Goll shows degeneration in some of its fibers on the same 
side up to the medulla, but the number steadily diminishes and 
is much less than that after section of the posterior columns of 
the cord. 
CEREBRUM. CEREBELLUM AND SEMICIRCULAR 
CANALS. 
The sigmoid gyrus of the dog's brain is exposed by trephining. 
This corresponds to the Rolandic area of the human brain. It 
curves about the cruciate fissure which corresponds to the fissure 
of Rolando (sulcus centralis) in more highly developed brains. 
Observe that stimulation of the sigmoid gyrus by a tetanizing 
current of suitable strength (i. e., the minimal current which 
will cause contractions) produces contractions of the muscles of 
the hind leg, fore leg, or neck, according to the part stimulated. 
The movements only occur on the opposite side of the body. They 322 
Laboratory Exercises in Physiology 
are not co-ordinated and may consist of irregular, spasmodic 
contractions of groups of muscles. By using a very strong stim- 
ulus spasmodic contractions of the same side of the body may 
be produced by diffusion in the spinal cord. 
After having localized the motor area by stimulation, this 
part of the cerebral cortex is removed by scooping out the corti- 
cal substance. The entire operation is done with surgical clean- 
liness and the wound is closed. As soon as the animal recovers 
from the anesthetic, note the temporary paralysis of the parts 
of the body whose cortical centers have been destroyed. 
Observe the animal for the next few days and note that the 
paralysis rapidly disappears. The movements of locomotion are 
soon regained and the "deficiency phenomena" are only seen 
in some unusual movements of the body. The animal is able to 
walk and run without any permanent paralysis. Such remark- 
able recovery only occurs in the lower animals, where the activities 
of the spinal cord depend upon the flow of the afferent impulses 
from the periphery more than the impulses from the cerebral 
cortex. 
224. The Effects of Removing the Cerebral Lobes in the 
Frog: The cerebral lobes in several frogs have been removed 
on the previous day. The optic tracts, optic lobes, medulla and 
spinal cord are intact. 
Observe (1) that the posture is the same as that seen in a 
normal frog and quite different from that of the "spinal" or 
reflex frog; (2) the croaking reflex of the male frogs is present; 
(3) the "balancing" reflex from gradually changing the base of 
support is preserved; (4) all the movements of the body are co- 
ordinated and performed as perfectly as in a normal frog so 
that no paralysis can be detected; (5) the frog avoids opaque 
objects in hopping; (6) not only is the frog able to maintain 
body equilibrium, but it swims perfectly when placed in water; 
(7) the frog does not move spontaneously, but only in response 
to some form of stimulus. 
225. The Effect of Removing the Cerebral Lobes in a 
Pigeon: The1' cerebrum of a pigeon has been removed two 
hours before the demonstration. Observe that (1) the appear- 324 
Laboratory Exercises in Physiology 
ance is different from that of a normal bird; (2) the animal 
balances perfectly on a perch and regains its balance when dis- 
turbed; (3) the animal walks and flies when compelled to do so, 
showing that it can maintain body-equilibrium and perform the 
most complex co-ordinate movements of which it is capable; (4) 
there is no spontaneity of movement and the animal remains 
quiet when undisturbed. 
325. The Effect of Injuring the Cerebellum in a Pigeon: 
The animal has had its cerebellum destroyed two hours before 
the demonstration. Observe that the dullness and inactivity 
which result from removal of the cerebrum are absent in this 
pigeon. The pigeon can perform complex movements and does 
so spontaneously, but co-ordination is disturbed and at times the 
animal has difficulty in maintaining body equilibrium. At times 
forced movements may occur, i. e., activities which seem to be 
beyond the control of the will. 
227. The Effects of Destroying the Semicircular Canals in 
Frogs: These are destroyed by introducing a pithing-pin into 
the temporal bone. Note that the frog can not maintain the 
normal posture, but turns the head towards the injured side. Ob- 
serve that these frogs can not hop or swim as normal animals do, 
but perform circular or spiral movements. Co-ordination of the 
movements of the body is completely lost. 
228. The Effects of Destroying the Semicircular Canals in 
Pigeons: (1) Observe the forced movements and lack of co- 
ordination in a pigeon which has had all the semicircular canals 
destroyed on one side. 
(2) Describe the. movements in a pigeon whose external (hor- 
izontal) semicircular canals have been destroyed on both sides. 
(3) Describe the movements and the way a pigeon falls after 
destruction of the superior or anterior vertical semicircular 
canal on one side and the posterior vertical canal of the oppo- 
site side. 
(4) Describe the effect of destroying the posterior vertical 
semicircular canals of both sides.