NOTES ON
PRACTICAL PHYSIOLOGY
\i\
JOHN MALCOLM. M.I).
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NOTES ON
PRACTICAL PHYSIOLOGY
For the use of Students of Medicine,
BY
JOHN MALCOLM, M.D. (Edin.),
Professor of Physiology In tlie University of Otago, and formerly
an Assistant to the Professor of Physiology In the
University of Edinburgh.
3llustrate&.
UUNEDIN :
FERGUSSON & MITCHELL Ltd.,
PRIXTERS L[TH0GRAPHERS and MANUFAC TURING SfATIOKERS
1909.
PREFACE.
This short description of the experiments usually performed
in classes of Practical Physiology has been written specially for
students attending the class of Practical Physiology at the
University of Otago.
The subject is treated in the order of the systematic lectures
to enable students to study the theoretical and practical sides
of the subject together.
Inasmuch as Physiology, besides being an excellent ground
for general scientific training, is a subject of direct importance
in Medicine, due attention has been paid to those methods of
clinical research that can be profitably pursued by medical
students of the second year, but it is almost unnecessary to say
that many of the experiments described are not directly applicable
to the problems of disease.
It would be difficult to give a list of all the works consulted
in the preparation of these exercises, but for the convenience of
students who wish to extend their knowledge, the author can
recommend the following books to which he himself is much
indebted, viz., the Practical Exercises in Stewart's Manual of
Physiology ; Milroj^'s Practical Physiological Chemistry ; Porter's
Introduction to Physiology ; and Practical Physiology by
Beddard, Edkins, Hill, Macleod, and Pembrey. For those who
can read German, Thierfelder's edition of Hoppe-Seyler's Hand-
buch der phys. u. path. chem. Analyse, and the Handbuch der
phys. Methodik edited by Tigerstedt, now appearing in parts,
are the best authorities on chemical and experimental work
respectively.
I am indebted to my friend, Mr. J. Brown, for the drawings
in perspective.
J.M.
University oe Otago,
dunedin, n.z..
May, 1909.
CONTENTS.
(The numbers given refer to the paragraphs).
CHAPTER I.
The Chemical Constituents op Tissues in General —
A. The Chemical Elements Found in the Tissues —
Inorganic (chlorides, etc.) (2-10)
Organic (carbon, etc.) (11-14)
B. The ''Proximate Principles" of 'the Tissues and
Food—
I. The Proteins —
A. Colour Reactions - (15-20)
B. Precipitation Reactions - (21-24)
C. The Action of Neutral Salts - (25-29)
D. Some Physical Properties of Proteins (30-32)
E. Action of Various Reagents on Protein (33-41)
F. Varieties of Protein.
II. The Carbohydrates —
A. Tests for Reducing Sugars in General (42-51)
B. Varieties of Carbohydrate important
in Physiology —
Monosaccharides (52)
Disaccharides (53-55)
Polysaccharides (56-71)
III. Fats (72-73)
OHAPTEE II.
The Animal Cell and Simple Tiss0ES—
A. Chemistry of protoplasmic cells and simple tissues (74-77)
B. Chemistry of Epithelial tissues (78-79)
C. Chemistry of Connective tissues (80-84)
CHAPTER III.
Muscle and the Action op Stimuli in General —
Extensibility and Elasticity of Muscle (85-92)
Action of Artificial stimuli in General (93-104)
The Contraction of Muscle (105-11.3)
The Chemistry of Muscle (114-127)
CHAPTER IV.
Nerve Tissue and Electro-Physiology.
Excitability and Conductivity—
The Influence of Galvanic Electricity
and Pfliiger's Law (128-135)
Fatigue, double conduction and velocity
of impulse (136-138)
Electro-Physiology (139-142)
CHAPTER V.
The Blood.
Enumeration of Corpusles (143-145)
Reaction, Specific Gravity, etc. - (146-149)
Coagulation (150-152)
Chemistry of Blood — Serum (153-159)
Hemoglobin (160-169)
CONTENTS. VU
CHAPTER VI.
Circulation or the Blood —
Anatomy and Histology - (170)
Physical. Action of the valves, frog's cardiogram,
heart sounds, and human cardiogram
(171-174)
The Pulse
(175)
Blood Pressure
(176-178)
Velocity of Blood Plow
(179)
Plethysmography
(180)
Vital. Automatism, conduction, etc.
(181-184)
Reaction to Stimulation
(185-190)
Cardiac Nerves in the Frog
(191-192)
Effect of toxic substances
(193-194)
Circulation in the vessels
(195-196)
CHAPTER Vn.
Ebspiration —
Respiratory Movements and Bounds (197-199)
Spirometry, Intrapulmonary Pressure (200-201)
Chemistry of Respiration and Blood (Jases (203-206)
Method of performing Artificial Respiration (207)
CHAPTER VIII.
Alimentary System—
Movements of the Alimentary Tract (208-209)
Chemistry of the Digestive Juices—
A. Saliva (210-215)
B. Gastric Juice - (216-226)
C. Pancreatic Juice - (227-231)
D. Bile - (232-243)
E. Succus Entericus (244-247)
Viii CONTENTS.
CHAPTER IX.
The Urine—
Reaction and Specific Gravity (248-249)
Chemistry
of the Urine —
A.
Organic Constituents
(250-266)
B.
Inorganic Constituents
(267-271)
C.
Pathological Constituents
- (272-282)
Cryoscopy
(283)
CHAPTER X.
Metabolism and Dietetics —
Glycogen in the Liver (284)
Phloridzin glycosuria - (285)
Analysis of Food Stuffs—
Milk and Cheese - (286-293)
Eggs (294-297)
Vegetable Food Stuffs (298-304)
Experiment on General Metabolism (305)
A. The water percentage of the food -
B. The percentage of ash
C. The percentage of protein
D. Estimation of fat in meal, etc.
E. Estimation of carbohydrate
F. Feeding of animal, etc.
G. Estimation of phosphorus
H. The carbon balance
I. The lime and magnesia balance
J. The chlorine balance.
Internal Secretions - - (306-310)
CONTENTS. IX
CHAPTER XI.
The Nervous System and Special Senses—
Central Nervous System —
(. Histology, Reflex Action and Reaction
Time - - (311-315)
The Special Senses —
Vision - (316-325)
APPENDIX. page.
List of Reagents and Apparatus required for class
work in general —
Chemical Room 168
Experimental Room 160
Special Apparatus, Material and Reagents required
for some of the experiments 160-168
Miscellaneous Instructions regarding the use of
chemical apparatus - 169
Comparison of metrical with British measures 170
Index - - - 171
LIST OF ILLUSTRATIONS.
^I*^- PAGE
1. Method of using crucible and lid 3
2. Apparatus for studying extensibility of muscle 29
3. The use of the lever stop 31
4. The crank myograph lever 34
5. Galvanic circuit with monocord inserted 36
6. von Fleischl's Rheonome 38
7. Simple stimulating arrangement - 41
8. Course of the current through Neef's hammer 43
9. Apparatus for taking a record of contraction of muscle 46
10. Exp. on the Genesis of Tetanus 51
11. Exp. on conductivity and excitability of nerve fibre 57
12. Exp. on the influence of the anode and cathode in
excitability of nerve 69
13. Apparatus for studying of Pfliiger's Law, etc. 61
14. The myograph fitted with a straight lever and frictionless
point for heart work - 81
15. Rat's cage for metabolism experiments 137
16. Estimation of CO^ (Haldane's method) 152
17. Measurement of simple reaction time for sight 154
CHAPTER I.
THE CHEMICAL CONSTITUENTS OF TISSUES
IN GENERAL.
(A) THE CHEMICAL ELEMENTS FOUND IN THE
TISSUES.
The chief constituents of dead cells are certain inorganic
salts, proteins, fats, carbohydrates, extractives and water.
THE INORGANIC CONSTITUENTS may be examined in the
ash of any cellular organ such as thymus, pancreas, muscle, etc.
To obtain the ash one must destroy the organic matter by
combustion or incineration in a crucible.
(1) Put some of the minced and dried tissue, about the bulk
of a walnut, in a porcelain crucible. Put on the lid and place the
crucible upright on a pipeclay triangle which is supported on a
tripod over a bunsen flame. Begin with a small flame and grad-
ually increase it till the whole mass is completely charred and all
bubbling has ceased. Allow the crucible to cool, extract the black
material with a few c.c. of distilled water previously heated to
boiling in a test tube, filter through an ash-free filter paper, and
test the filtrate for chlorides (2). Meanwhile, place the filter-paper
with its contents in the crucible and, leaving the lid ofE, re-apply
the flame, at first gently to drive ofi the moisture ; when dry,
increase the flame and incinerate completely, placing the lid of
the crucible in such a way as to direct a current of heated air over
the contents, as shown in Fig. 1,
2 Peacttcal Physiology.
"When no more black carbon remains, cool the ash and extract
it with 10-20 e.c. of hot distilled water containing a small
quantity of HCl. Note the efiervescence which sometimes
occurs on adding this, due to the presence of carbonates. Filter
and use the filtrate for tests (3), (4), (6), (7), (8), (9), (10).
(2) Chlorides. — Addition of silver nitrate gives AgCl as a
white, curdy precipitate insoluble in nitric acid, soluble in
ammonia.
(3) Phosphates. — Ammonium Molybdate solution and nitric
acid give a yellow precipitate of phospho-molybdate of ammonia,
more marked when the mixture is heated nearly to boiling point.
(4) Uranium nitrate and sodium acetate solution give a
whitish precipitate when heated. The precipitate is soluble in
free nitric acid [see later under estimation of phosphates (269) ].
(5) Carbonates.- — COg results from the combustion of the
organic matter and forms carbonates if sufficient alkali is present.
This occurs rarely in animal, though usually in vegetable tissues.
Effervescence on adding HCl indicates its presence.
(6) Sulphates. — Sulphates arise in a similar way from oxida-
tion of the organically bound sulphur of proteins. Barium chlor-
ide and HCl give a dull white precipitate. HCl is added to keep
in solution the barium phosphate which would otherwise be
precipitated.
(7) Sodium may be detected by the flame test ; and
(8) Potassium by addition of sodium-hydrogen-tartrate —
fine crystalline precipitate. (N.B. — The solution must first be
neutralised).
(9) Calcium. — When testing lor calcium in an acid fluid such
as this, ammonia must first be added till the reaction to litmus
is slightly alkaline ; then render the fluid slightly acid with acetic
or ci1;ric acid, and add ammonium oxalate — a precipitate of cal-
cium oxalate forms.
(10) Magnesium.— Filter off the precipitate of calcium
oxalate obtained in (9) and make the filtrate strongly alkaline
with ammonia. Since phosphates are practically always present,
Practical Physiology.
Fig. 1.
Method of using the crucible an J lid when
completing incineration.
4 Practical Physiology
a precipitate of triple phosphate (MgNH^PO^) forms if there
is sufficient magnesium.
Iron is also present in the ash of most tissues, but is especially
abundant in the red blood corpuscles (see 160 and 296).
Of the foregoing, the chlorine, fhosfhorus, sodium, potassium,
calcium and magnesium form tlie main bulk of the inorganic
constitutents.
THE ORGANIC CONSTITUENTS, on the other hand, are
carbon, oxygen, hydrogen, nitrogen, sulphur, phosphorus, with
occasionally other elements such as iron, iodine, fMorine, etc.
On complete incineration of the tissue many of these form volatile
products such as COj, HgO, ^0^, and are therefore driven off,
unless sufficient alkali is present.
The presence of carbon is sufficiently indicated by the charring
which occurs on incineration.
(11) Nitrogen (Lasseigne's test). — Place in a dry test tube a
small amount of the dried tissue such as mince, add a small piece
of metallic sodium, hold the end of the tube in the flame till it be-
comes red hot and then plunge it into a small quantity of dis-
tilled water in a porcelain basin ; the tube breaks and its contents
partially dissolve in the water. Filter, and test some of the
filtrate for the presence of a cyanide by adding a few drops of
weak ferrous sulphate solution, acidifying with HCl, and
then adding a ferric salt, such as ferric chloride : — A pre-
cipitate of Prussian Blue forms if a cyanide is present, and
indicates the presence of nitrogen in the material originally taken.
(12) Sulphur may also be tested for in the same filtrate.
Add sodium nitro-prusside — a reddish-violet colour indicates the
presence of sulphur as a sulphide.
(13) Another method of demonstrating the presence of
sulphur or of phosphorus is to incinerate the tissue in a porcelain
crucible with the addition of a considerable amount of dry sodium
carbonate. To hasten the oxidation small quantities of pot-
assium nitrate may be added from time to time. After coolinp
dissolve the residue in distilled water and test for sulphates (6J
and for phosphates (3).
Practical Physiology. 5
(14) Hydrogen and Oxygen are shown to be present by
heating the thoroughly dried material in a dry test tube. Watery
vapour forms and condenses in the upper part of the tube.
Iodine occurs in appreciable amount in the thyroid gland
(see test 310).
(B.) THE "PROXIMATE PRINCIPLES" OF THE TISSUES
AND FOOD.
The elements just mentioned (C, 0, H, N, S, P, ) are com-
bined with one another so as to form definite molecules of organic
substances which can be isolated in more or less pure form.
They are the frnteids (or proteins), carbohydrates, and fats.
The tissues also contain small quantities of substances derived
from these, such as creatin, xanthin, lactic acid, lecithin, choles-
terin, etc. These are generally spoken of as " extractives."
(I) PROTEINS or PROTEIDS).— These have certain colour
reactions in common, and the majority are precipitated by
certain reagents, but there are individual differences which,
along with our growing knowledge of the constitution of
the protein molecule, form the basis of the classification.
(A) Colour reactions for Proteins in general.
(15) Xanthoproteic Test. — To a solution or suspension of
protein in water add about an equal amount of nitric acid. Heat
to near boiling, cool under the tap, and add ammonia cautiously
to the upper part of the solution. If a yellow colour appears on
adding the ammonia, or if a yellow colour is produced by heating
with the nitric acid and is darkened to orange by the alkali-^the
result is positive. The presence or absence of a precipitate on
adding the nitric acid is to be disregarded.
Apply the test to the following proteins if available : egg-
white, gelatine, albumose, keratin, and make notes of the result
in each case.
(16) MillorCs Reaction. — Millon's reagent is prepared by
dissolving metallic mercury in strong nitric acid and then diluting
with water. It consists of a mixture of mercurous and mercuric
6 Practical Physiology.
nitrates in nitric and nitrous acid. Add some of the reagent to
the protein solution (a precipitate may or may not appear).
Heat the mixture. A brick-red colour indicates a positive result.
Apply the test to the proteins mentioned above and to 5%
carbolic acid (phenol) and note the results.
(17) Biuret Reaction (or Piotrowski's Test). — To a pro-
tein solution add one drop cuprio sulphate solution and then
some KOH sufficient to make strongly alkaline. A fine violet or
pink colour develops if the result is positive. If the result is
doubtful add a little more cupric sulphate cautiously, but avoid
excess of copper and do not mistake the blue colour of a solution
of cupric hydrate in the alkali for the fine colour of the reaction .
Kepeat the test with the proteins available and compare test (252)
on the substance biuret itself.
(18) Glyoxylic or Hopkins' Reaction. — To the protein solu-
tion add an equal amount of glyoxylic acid solution ; mix and
add concentrated sulphuric acid so as to form a layer at the
bottom of the tube. A bluish violet colour develops at the plane
of contact (this may take some time to appear).
(19) Liebermann's Test. — To some concentrated hydrochloric
acid in a test tube add one drop of undiluted white of egg — heat,
at first gently then up to boiling — a violet colour appears.
(20) Molisch's Test. — To a protein solution add a few drops
of a solution of alpha-naphthol in methyl alcohol. Mix, and add
some concentrated sulphuric acid. A reddish to violet colour
develops at the plane of contact.
The last three tests are not generally used for the detection
of proteins. Tests (15), (16), (18), and (19), are due to the pre-
sence of an aromatic radicle in the protein molecule and are there-
fore weak or negative with gelatine solations and some other
proteins which have little or no aromatic radicle. Tests (19),
and (20) are due to a carbohydrate radicle, and test (17) to a
peculiar grouping of the atoms which is found in practically all
proteins.
Practical Physiology. 7
(B) Precipitation Tests for Proteins in general.
(21) Coagulation hy Heat may be included under these.
Heat some egg white solution and note clouding and formation
of a coagulum ; this result is facilitated by the presence of
neutral salts and a faintly acid reaction. In the absence of
these two conditions a dilute solution of protein may become
opalescent but does not form a distinct coagulum. Test this
statement with a very dilute egg white solution as follows : — Into
each of two clean test tubes put about the same quantity of the
diluted protein given you ; to one add a little sodium chloride ;
heat both tubes to boiUng and note the difference between them.
The influence of the reaction may be tested in a similar way.
The temperature at which a coagulum forms is distinctive
of some varieties of protein, see (32). Apply the heat test to solu-
tions of the following and note the results — egg-white, globulin,
hemoglobin, gelatine, albumose, caseinogen.
(In testing urine for albumin a few drops of acetic acid
should always be added to the urine after heating it to boiling.
This serves the double purpose of facilitating coagulation and
redissolving the precipitate of earthy phosphates which some-
times appears on heating that fluid).
(22) Mineral Acids. — To some egg white solution add a few
drops of 20% mineral acid (nitric, sulphuric, or hydrochloric) a
white precipitate appears in the cold. The ' ' ring ' ' method of
application is useful here — hold the test tube containing the
protein solution at an angle of about 45° to the vertical and pour
the acid slowly down the lower side of the tube. It falls to the
bottom, and on holding the tube upright the two fluids will
be found one above the other with a white ring or disc of pre-
cipitated protein on the surface of the acid.
(Heller's test consists in the application of nitric acid in this
wav. It is very frequently used in testing urine).
Kepeat the tests with gelatine, caseinogen, and albumose.
(23) Salts of the Heavy Metals. — The majority of these com-
bine with and precipitate proteins from their solutions, e.g.,
mercuric chloride, copper sulphate, ferric chloride, etc. One test
8 Practical Physiology.
used clinically may be put under this heading — acidify the
solution of protein with acetic acid and add a few drops of pot-
assium ferrocyanide, a precipitate forms if albumin or globulin
is present, if only traces of these are present the precipitate may
take several hours to develop. Gelatine gives this reaction if
the solution is not too concentrated.
(24) Other Preci'pitants. — There are many other good pre-
cipitants of proteins which are used as qualitative tests, and some-
times for quantitative estimation (in Esbach's tube), sometimes
also they are used to rid a solution of all protein matter. Some
of these are tannic acid, ficric acid, trichloracetic acid, salicyl-
sulphonic acid, potassio-mercuric iodide with hydrochloric acid, etc.
Test as many of these reagents as are available. Alcohol is also
used in the same way. When the precipitate first forms it can
be redissolved in water, but after long contact with the alcohol
it becomes insoluble in water, i.e., coagulated.
This illustrates the difierence between the terms ' ' precipita-
tion" and "coagulation" of proteins.
(C) The action of the Neutral Salts on Protein.
These have an important action on proteins in solution, and
they are used to test for the presence of a protein by precipitation
but to a larger extent to remove one or all proteins completely
from solution. The precipitated protein can be recovered un-
changed from the precipitate, so that the action is more physical
than chemical.
The salts are used in the following ways : —
(a) Full Saturation. — For this it is necessary to add the solid
crystals of the salt to the protein solution and shake till no more
crystals dissolve. The fluid must not be heated if it contains
proteins which are coagulable thereby, otherwise it may.
(b) Half Saturation. — For this a fully saturated solution of
the salt is required and is mixed with an equal quantity of the
fluid to be treated. The result, on thorough mixing, is half satura-
tion of the whole fluid. This half (or 50%) saturation is the
degree of partial saturation most commonly employed, but the
solubility of proteins in partially saturated solutions of salts has
Practical Physiology. 9
been more carefully worked out, e.g., if we take serum-globulin
solution and add fully saturated ammonium sulphate solution,
precipitation of the globulin begins when the mixture contains
'^9% of the saturated salt solution, and becomes more and more
marked as the percentage of salt solution increases till 46%
is reached, at which point all of this globulin is thrown down, so
that in this case half saturation (50%) is more than enough to
precipitate all the substance. " The limits of concentration
necessary to initiate and to complete precipitation of different
proteins are as characteristic of each special protein as is the
solubility of a crystalline substance." (Cohnheim).
Test the truth of the following statements with the reagents
and material available : —
(25) Ammonium Sulphate in full Saturation precipitates all
proteins except peptones. — To some egg white solution add crystals
of ammonium sulphate and shake vigorously till no more of the
salt wiU dissolve. A precipitate forms — filter and test the filtrate
for protein (15 or 16) no result — because egg white contains no
peptone.
Repeat the test on an albumose and peptone mixture. A
precipitate of albumose forms and the filtrate gives a positive
result when tested for protein by (17).
[In applying the biuret test to a fluid containing an ammonium
salt, sufficient alkali must be added to displace the ammonia,
therefore make a very concentrated solution of KOH or NaOH
by dissolving some of the solid alkali in a small quantity of water,
cool, and add this instead of the ordinary KOH.]
Repeat with a gelatine solution and note how the precipitate
frequently floats up to the surface of the heavy solution of Am.
SO4.
(26) Ammonium Sulphate in half saturation precipitates
globulins, primary alhumoses, caseinogen, and some other proteins. —
To some 1 in 5 egg white solution add an equal amount of satu-
rated AmSO^^, a precipitate of egg globulin forms, filter and test
the filtrate for egg albumin by (15), (21), or other test, e.g.,
full saturation with AmSO^ (25). Repeat the half -saturation
with a mixture of albumoses (primary and secondary). Filter
10 Practical Physiology.
and test the filtrate in this case by some of the colour tests (15),
(16), or (17) ; the secondary albumose which is present is not
precipitated by the half-saturation with AmSO^ and is found in the
filtrate.
Eepeat the half saturation with caseinogen and any other
protein available.
(27) Sodium Sulphate in full saturation at a temperature
of 37°C acts in the same way as full saturation with AmS04 at
room temperature.
Test this on egg white solution using the water bath to warm
the mixture.
(28) Magnesium Sulfhale in full saturation at room tempera-
ture acts in the same way as half-saturation with AmSO^.
Test this as ia (2fi). The solution must be shaken for a con-
siderable time with the MgS04 to ensure full saturation.
(29) Sodium Chloride in full saturation precipitates all
globulins, caseinogen, and nucleo-proteins.
It may be noted here that while certain proteins are precipit-
ated by concentrated solutions of salts, a small precentage of
neutral salt is required to keep some proteins in solution, especially
the globulins, and it is chiefly sodium chloride which seems to play
this part in the body.
(D) Some Physical Properties of Proteins.
Indiffusihility. — Most proteins are unable to diffuse through
animal or parchment membranes, i.e., they belong to the class of
bodies known as colloids.
(30) Place some blood serum (1 in 5) in a parchment tube,
tying one end securely to form a test tube of it, and suspend it in
a jar of water. After a few days test the water in the jar. It will
be found to contain most of the salts, e.g., chlorides, but no
protein. The tube will contain the proteins and some
of the globulin (eu-globulin) is seen to be precipitated due to
removal of the salts.
Filter some of the contents of the dialyser, and half-saturate
with AmS04 — a precipitate (pseudo-globuhn) will appear.
Practical Physiology. 11
In spite of their colloid nature some proteins are ori/stalKsable
e.g., Hsemoglobin.
(31) To a drop of rat's or of guinea pig'a blood on a slide
add a similar amount of distilled water, cover and allow the fluid
to evaporate slowly at room temperature. Crystals of Hfemo-
globin appear at the edges — note their shape. Some proteins
- appear naturally in crystalline form in seeds, and others can be
crystallised by partial saturation of their solutions with ammon.
sulphate and adding acetic acid till a precipitate just begins to be
visible. Examine crystals of serum albumin of horse's blood
which have been made in this way.
(32) Temferature of Heat Coagulation. — The point at which
this occurs varies with the amount of protein in solution, the salts
present, and the degree of acidity. The more marked these con-
ditions are, the lower is the temperature of coagulation, but in
spite of these difficulties the temperature of coagulation is of
value in recognising some classes of proteins, e.g., those found in
muscle.
Make the fluid (e.g., egg white 1 in 5) faintly acid with acetic
acid, put a thermometer in the test tube, and place this within
a second larger test tube half full of water, and then put the whole
into a water bath heated by a moderate-sized flame. Allow the
temperature to rise very gradually, stirring the protein solution
occasionally with the thermometer. Note the position of the
mercury when distinct cloudiness occurs ; try to keep the fluid
at this temperature for a little till a distinct flocculent coagulum
forms. Filter into a fresh tube and repeat the experiment with
the filtrate ; another coagulum may be obtained at a higher temper-
ature than the first. Since egg white is a mixture of several
proteins, coagula may occur at 50° — 60°, at 64° (albumins) and
about 75° (ovo-globulin).
(E) Action of various reagents on Proteins.
Alkalis. — Strong alkahs (NaOH or KOH) added to concen-
trated protein solutions cause swelling and formation of a jelly
of alkah-albumin (Lieberkuhn's Jelly).
(33) Make this jelly by adding 23% NaOH to undiluted egg
white. If this be now diluted with an equal amount of water and
heated, or
12 Practical Physiology.
(34) If 10 % KOH be added to egg wMte and heat applied,
there results a decomposition of the proteins and ammonia is
evolved — test for this by holding moist red litmus paper over the
mouth of the test tube.
(35) If the contents of the tube be now cooled and a few
drops of dilute sodium nitroprusside added, a reddish violet colour
appears indicating the presence of a sulphide.
(36) Or the sulphide may be tested for by the addition of
neutral lead acetate (black ppt.). This shows that the action of
strong alkaUs and heat on proteins is a very penetrating one,
some nitrogen is spht off as ammonia and some sulphur which
combines with the alkali to form a sulphide ( ' ' loosely combined
sulphur "). Less energetic action of heat (4:0°C) and weak alkali
cause formation of alkali- albumin as shown in the next experi-
ment.
(37) To some 1 in 5 egg white add several drops of 10% KOH.
and place in the water bath at 40° C. for ten to twenty minutes.
The proteins become changed into alkali-albumin.
(38) Test this solution as follows : — boil — no coagulation
results ; neutralise with acetic acid after adding a drop of litmus
solution to act as indicator — a precipitate comes down which
dissolves in excess of the acid.
(39) Acids. — Concentrated acids also cause formation of a
jelly when acting on concentrated protein solutions. To some
undiluted egg white add some glacial acetic acid — a jelly results
which is more opaque than Lieberkuhn's.
(40) To some 1 in 5 egg white add some 10% acetic acid and
keep at 40° C. (as in 37 above) for twenty minutes — acid-albumin
is formed.
(41) Test this in similar fashion to (38) : — boil — no coagula-
tion ; add litmus solution and neutralise — acid-albumin is thrown
down and redissolves in excess of the alkali.
On the whole the action of acids and heat on protein is less
penetrating than that of alkaUs, but the result of long continued
action is the same — the meta-protein is converted into proteoses,
Practical Physiolouy. 13
peptones, and finally into the amido acids, hexone bases, ete.,
which are the ultimate decomposition products of proteins.
Action of Ferments. — Two classes of ferments act on pro-
teins : —
(a) Some proteins are coagulated by specific ferments, e.g.,
cascinogen (by rennin), fibrinogen (by fibrin ferment), myosinogen
and paramyosinogen (ferment doubtful).
(b) All proteins are split by the digestive ferments pepsin,
trypsin, erepsin, into their meta-protein derivatives and ultimately
into the amido acids, etc.
These wUl be studied in connection with milk, blood, diges-
tion, etc.
(F) Varieties of Proteins.
(a) Protamines.
(b) Histones.
(c) Albumins, found free in nature, e.g., egg albumin (present
in egg white) serum albumin of blood, lact-albumin, etc. These
give all the typical protein reactions and can be isolated from
globulins by neutral salts.
{d) Globulins generally occur along with albumins, are in-
soluble in distilled water (except the pseudo globulins)
and insoluble in concentrated solutions of neutral salts.
Repeat experiments (26) and (28) with an egg white or serum.
(e) /Sckro-protems (albuminoids) e.gr., collagen gelatin, keratin,
etc. See under Connective Tissue, tests (81), (82), (78).
(/) Phospho-'proteins (nucleo albumins), e.g., caseinogen,
vitellin, see under milk and eggs.
(g) Conjugated proteins, or protein united to a prosthetic
group, e.g., nucleo-protein (nuclein -\- protein), gluoo-protein such
as mucin (protein -|- a carbohydrate), chromo-portein such as
haemoglobin (protein -f- hsematin).
(h) Derivatives of Protein.
(1) Meta-proteins or albuminates, e.g., acid and alkali
albumin, Cu- Fe- and Hg-albuminates.
14 Practical Physiology.
(2) Proteoses or albumoses.
(3) Peptones.
(4) Polypeptides.
These varities of protein will be considered under the different
tissues and fluids, and under digestion.
11. CARBOHYDRATES. — The chief carbohydrates found
in the body are glucose, lactose, maltose, glycogen, and
dextrin. Cane sugar and starch are important food stuffs, and
will also be considered here.
(A) Tests for reducing Sugars in general. — Glucose or
dextrose is the aldehyde of a hexatomic alcohol. Owing
to the presence of the aldehyde group, CHO, it can absorb
oxygen from substances capable of yielding it and so form acids,
COOH. This occurs most readily in hot alkaline solutions,
and the newly formed acids unite with the alkah. Consequently
all ' ' reduction ' ' tests are done in alkaline solution [except
Barfoed's test, (44)],
(42) Trommer's Test. — To some glucose solution add some
KOH and a few drops of cupric sulphate solution. Flakes of
cupric hydrate appear, which are dissolved by the glucose to
form a blue solution. Continue adding the CUSO4 till no more
flakes will dissolve and then heat. A yellow coloured ppt. which
changes to red appears just below boiling temperature. This
indicates "reduction," the cupric hydrate, Cu(0H)2, being
reduced to cuprous hydrate, Cu2(0H)2, and from this red
cuprous oxide, CugO, separates out. In applying the test to
some solutions, e.g., starch, black flakes of cupric oxide (CuO)
may appear — this of course does not indicate reduction.
(43) FehUng's Test.—
[Solution A., 34.64 grm. crystals of cupric sulphate in 500 c.c.
water ; solution B., 173 grm. sodium potassium tartrate -}- 60
grm. sod. hydrate in 500 c.c. water. Mix exactly equal amounts
of A. and B. If kept for some time Fehling's solution may reduce
of itself on boiling, therefore test before use.]
Practical Physiology. 15
Take some Fehling's solution in a test -tube, heat to boiling.
If no reduction occurs add about an equal amount of the solution
to be tested and heat again to boiling — if glucose is present in
sufficient amount reduction appears just at or before boiling
point. In this case the red oxide frequently separates out more
readily than in Trommer's Test.
If ammonia is added to the Fehling's solution in sufficint
amount, it holds the reduced oxide of copper in solution so that
the result is a clear colourless fluid which gradually deposits Cu^O as
the ammonia boils off (cf. Pavy-Fehling 276b).
(44) BarfoerTs Test (cuprio acetate in acetic acid solution).
This is the only reduction test commonly employed where the
reaction is acid. Glucose is able to reduce it. Mix equal quan-
tities of the reagent and the carbohydrate solution and heat to
boiling point, but do not prolong the boiling for more than one
minute. Cuprous oxide appears when the result is positive.
Maltose and lactose give negative results with this test, and
even with glucose the reduction is not very marked. Apply the
test to solutions of these three sugars.
(45) Boettger's Test. — Add a little bismuth subnitrate
powder to the glucose solution and about double that amount of
dry sodium carbonate. Boil for a short time. A black or grey
deposit indicates reduction of the bismuth salt.
Nylander's Solution is sometimes employed instead of the dry
bismuth subnitrate. It bears the same relationship to Boettger's
Test that Fehling's does to Trommer's.
(46) Ammoniacal Silver Nitrate Solution gives a mirror of
reduced metallic silver on heating with a reducing sugar.
(47) Several organic pigments are reduced and show colour
changes or loss of colour when heated with glucose in alkaline
solution. To some saffranin solution in a test tube add several
drops of KOH and some of the solution to be tested ; mix, heat to
boiling — the red colour changes to a yellow if a reducing sugar is
present. Cool, shake up with air, and note return of the colour.
16 Practical Physiology.
Repeat the test with, indigo-carmine solutioii. In many of
the reduction tests the substance reduced may be re-oxidised
by shaking with air or oxygen. This occurs easily in the fore-
going teat, and it can also be shown with the copper reductions
if the test tube is allowed to stand tUl next day. The oxidised
sugar is unaffected.
(48) Moore's Test. — Heat the glucose solution with some
caustic alkali, a yellow to brown colour appears due to forma-
tion of caramel. This test is given by all reducing sugars.
(49) Phenyl-hydrazine Test (Neumann's Method). — Mark the
level of 3 cc. on a large wide test tube by running in that
amount of water from a burette. Empty the tube. Put
into it 5 cc. of the suspected fluid, 2 cc. of 50% acetic
acid saturated with sodium acetate, and two drops of fluid
phenyl-hydrazine. Heat the mixture on the open flame
and keep it nearly boiling until the volume of fluid is reduced to
3 cc. Cool, examine the deposit with a microscope for yeUow
needle-like crystals of the osazone arranged in feather or double
fan shapes. If the deposit is amorphous heat again to boihng point
and allow to cool slowly. The crystals of the various osazones
(phenyl-glucosazone, phenyl-lactosazone, phenyl-maltosazone,
etc.,) are recognisable under the miscrosoope, but more exactly
by determination of the melting point of the purified crystals.
The older method of applying the test is to add a pinch of crystall-
ine phenyl-hydrazine hydrochloride and double that amount of
sodium acetate and heat on a boiling water bath for half an hour
or more. Crystals of the osazone separate out on cooling.
(50) Fermentation Test. — Add some baker's yeast (or cake
yeast which has been soaked in water to expel entangled air) to
the glucose solution and place the mixture in some form of tube
which will allow of the detection of gas formation (Southall's ureo-
meter or a special fermentation tube). Keep in a warm place,
preferably at 40° C, for several hours, and examine for gas (COg)
formation. Two control experiments should also be set on, one
with yeast and a fermentable sugar (glucose) to prove whether
the yeast is active, and one with yeast and water to see whether
the added yeast contains enough adherent sugar to cause gas
formation on being fermented.
Practical Physiology. 17
(51) The Polarimeter is used to detect the presence of
optically active substances such as sugars, and also to estimate
the amount present where the nature of the sugar is known.
The solution must be sufficiently transparent to allow light
to penetrate the whole length of the tube, and for this it is gener-
ally necessary in the case of urine to add some solid lead acetate,
shake thoroughly, and filter through a dry filter paper into a
dry vessel.
The method of manipulation depends on the form of polari-
meter used.
In using Fleisohl's Spectro-'polarimeter direct the instrument
towards a bright part of the sky, place the ' ' nuU-punkt ' ' or
empty tube in the body of the apparatus and examine the field
through the eyepiece. Two spectra, each crossed by a dark band,
will be seen, one above the other. Turn the analyser till these two
are exactly over each other and read the zero point, which may or
may not coincide with the zero of the scale. Repeat several times
and take the average. Now substitute for the empty tube the
tube filled completely with the solution to be tested and examine
the field again. If the bands have shifted one past the other the
substance is optically active, and according as the analyser has
to be turned to the right hand or to the left to make the bands
coincide, the substance is respectively dextro-rotatory or laevo-
rotatory. When the bands are exactly superimposed the per-
centage of glucose present can be directly read off on the scale in
this form of polarimeter. Use the vernier to get the first decimal
place, and take the average of several readings.
(B) Varieties of Carbohydrates important in Physiology.
I. Monosaccharides with the general formula CgHtjOe
(= hexoses) and C^H, qOj (= pentoses).
(a) Glucose has been already studied (42) — (51).
(b) Laevulose occurs in the alimentary tract from inversion
of cane sugar in food. It gives results similar to
glucose, with tests (42 — 50)., but rotates the plane of
polarised light to the left.
18 Practical Physiology.
(52) On heating a laevulose solution with an equal amount
of HCl and a few grains of resorcin, the fluid becomes a deep red
and a brownish red ppt. separates out.
(c) Galactose occurs along with glucose as a result of the
inversion of lactose.
(d) For Pentoses see under urine (278).
II. Disaccharides, general formula CjaHggOn.
(a) Cane Sugar. — A food substance. Does not reduce if
pure, try any or all of tests (42 — 48). Forms no
osazone (49).
(53) On boiling a solution of cane sugar with a few drops
of HCl, or under the influence of inverting ferments at body
temperature, cane sugar splits into dextrose and laevulose, and
the fluid will now give all the tests for these. Eepeat the reduc-
tion tests and (52) on this solution. Eemember to neutraUse
the HCl present before applying the reduction tests.
(54) Concentrated sulphuric acid when applied by the ' 'ring"
method causes a black (charred) band to appear at the plane of
contact with the cane sugar solution.
(55) Cane sugar slowly ferments with ordinary yeast. This
is an indirect effect due to an inverting ferment in the yeast which
splits the sugar into monosaccharides ; these then undergo
alcoholic fermentation with development of CO.^.
(b) Maltose. — A di-saccharide resulting from the digestion
of poly-saocharides such as starch. It gives positive results with
all the reduction tests, but is slower and less powerful in this
respect than glucose. It does not give Barfoed's Test (44). It
forms phenyl-maltosazone (49) the crystals of which are coarser
than those of glucosazone. It ferments easily (50). Test the
truth of these statements on a solution of maltose. On inversion
one molecule of maltose yields two of glucose.
(c) Lactose. — A di-saccharide found in milk. It gives all
the reduction tests except Barfoed's. With phenyl-hydrazine
it forms lactosazone, the crystals of which are finer and more
Practical Physiology. 19
closely matted together into balls than those of glucosazone are.
It does not ferment with ordinary yeast. On inversion it splits
into glucose and galactose.
III. Polysaccharides — general formula (CgHjoO^)" .
(a) Starch. — The chief form of carbohydrate in food. "
(56) Examine microscopically starch grains in a scrap-
ing from potato or in wheat, flour, etc.
(57) Add some powdered starch to cold water and note its
imperfect solubility. Heat to boiling ; an opaque or opalescent
appearance results. The grains burst and swell up forming a
mucilage or imperfect solution. Cool and use this mucilage for
tests (58) (62).
(58) Iodine test. — To starch mucilage add iodine solution
(dissolved in KI) a deep blue colour results which appears black if
concentrated starch mucilage is used, so that to see the blue colour
it may be necessary to dilute with water. This blue iodide of
starch is dissociated by heating and re-forms on cooling if all
the iodine has not been driven off. Alkah, if added, combines
with the iodine and a colourless solution results : the test must
therefore be done in the cold and in neutral or acid solution.
(59) Pure starch solutions do not reduce cupric hydrate.
do Trommer, Fehling or other reduction test.
(60) Tannic Acid precipitates starch.
(61) Starch is colloid, does not diffuse through a parchment
dialyser and is precipitated by neutral salts. Fully saturate
a starch solution with ammonium sulphate. Note precipitate.
Filter and test filtrate with iodine solution.
(62) On hydrolysis starch mucilage yields reducing sugars.
(a) Add some hydrochloric acid to starch mucilage and
boU for several minutes — the solution becomes clear ;
cool, neutralise the acid present and test for a
20 Practical Physiology.
reducing sugar. The starch, has been converted into
dextrins, maltose, and this again into glucose.
(&) To starch mucilage add some saliva and keep in the
water bath at body temperature (37°C) for a few
minutes. It will now reduce Cu(0H)2 owing to the
" presence of maltose (and dextrins).
(b) Dextrins. — A series of bodies with the same general
composition as starch, but of smaller molecule.
The two chief varieties are Erythro-dextrin and Aohroo-
dextrin. They result from the hydrolysis of starch aud gly-
cogen by acids or digestive ferments and the steps in the process
are as follows : — The starch is first converted into amiduKn, or
"soluble starch" ; this is shown by the previously opalescent
mucilage becoming clear while at the same time iodine gives a
blue colour with the solution ; erythrodeodrin is next formed and
the fluid now gives a red colour with iodine ; achroo- dextrin then
results and iodine now gives no colour reaction ; last of all the
sugars are formed. If the hydrolysis has been carried out with
diastatic ferments (ptyalin, amylopsin, etc.) maltose is the end
product, but if HCl is used the maltose is converted further
into glucose. It ought to be mentioned, however, that some
maltose appears early in the hydrolysis, that it is accompanied
by traces of another disaccharide, iso-maltose, and that in di-
gestion experiments in vitro it is difficult to convert the whole of
the dextrins into the maltoses unless these products are removed
as they are formed.
Test some commercial dextrin as follows : —
(63) Add iodine — note the reddish violet to reddish brown
colour — test the effect of adding KOH to this and of heating as
in (58).
(64) Dextrin solutions reduce cupric hydrate (43).
(65) Hydrolise some dextrin solution (62a) and note the
increased reduction power.
(66) Pasic lead acetate gives no precipitate with dextrin
solutions.
Practical Physiology. 21
Note also the smell and taste of dextrin.
The different varieties of dextrin can be isolated by their
insolubility in alcohols of different strengths and by precipi-
tation with neutral salts.
(c) Glycoqen. — "Animal Starch" found in liver, muscles,
leucocytes and other tissues of the body. It forms an opalescent
solution in water.
(67) Pure glycogen solutions do not reduce. Do Fehling's
or Trommer's test.
(68) On hydrolysis with weak HCl as in (62a) — glucose
results — Eepeat the test for reduction employed in (67).
(69) Iodine solution gives a mahogany red colour with gly-
cogen solutions which behaves like iodide of starch, when heated
or if the reaction is changed (58).
(70) Glycogen is completely precipitated when its solution
is saturated with ammonium or magnesium sulphate. This
serves to distinguish it from Erythro-dextrin, which is not com-
pletely precipitated. Compare the results of saturating a dex-
trin solution and a glycogen solution with AmSO^. In the
one case the filtrate gives a red reaction with iodine, which does
not occur in the ca^e of glycogen.
(71) Basic lead acetate precipitates glycogen but this can-
not be depended on as a distinction between it and dextrin.
in. FATS. — Fats are combinations of glycerine and fatty
acids the chief varieties of which are palmitic, stearic, and oleic.
These occur in varying proportions in the fat of the body and
food, and these variations account for their differences in fluidity —
the more olein there is, the softer is the fat and the lower its
melting point.
The fatty acids may be tested for by saponification of the fat.
(72) Saponification. — To some melted fat in a test tube add
an excess of caustic potash in alcohol, and keep the mixture in
22 Pbactical Physiology.
a boiling water bath till all the oiliness has disappeared.
The contents of the tube should now be perfectly miscible with
water for, by the action of the alkali and heat, the fat has taken
up water and has split into glycerine, and fatty acid which unites
with the alkali to form a soap.
Add some of the solution to water in a test tube and shake —
a froth forms.
Add some of the solution to an excess of warm 20% STil-
phuric acid in a test tube. The soap is decomposed and the free
fatty acid which results floats to the top in the form of oily glo-
bides which harden on cooling, and then resemble wax.
Add some of this to caustic potash, it dissolves, again forming
a soap.
(73) The glycerine part of the fat may be tested for by the
Acrolein reaction. To some fat in a dry test tube add more than
the same bulk of potassium bisulphate crystals and heat strongly.
A vapour which is very irritating to the nose and eyes is given
off. The cause of the irritation is a volatUe aldehyde, acrolein.
Like other aldehydes this is a reducing substance and if a piece
of filter paper moistened with ammoniacal silver nitrate is held
in the vapour it is blackened, cf. test (46).
CHAPTER II.
THE ANIMAL CELL AND SIMPLE TISSUES.
Slides illustrating the structure of typical cells and simple
tissues are to be examined at this stage, and experiments on
amoeboid and ciliary movement performed (See ScMfer's Essen-
tials of Histology).
(A) CHEMISTRY OF PROTOPLASMIC CELLS AND SIMPLE
TISSUES.
When extracts are made of very cellular organs such as the
thymus gland, pancreas, lymphatic glands, the most character-
istic substances obtained are nucleo-proteins.
(74) Minced thymus gland or pancreas is to be extracted
over night with ammoniacal water, then strained several times
through flannel. From this solution the nucleo-protein may
be precipitated by the cautious addition of acetic acid ; avoid
excess of acid as the precipitate is soluble therein. Allow the
nucleo-protein to settle, pipette off the supernatant fluid, add
some artificial gastric juice to the deposit, and place it in an in-
cubator at 40°C to digest.
Next day an insoluble residue of nuclein will be found —
draw some of this up in a pipette and note that, like the nucleo-
protein itself, it dissolves in alkalis and is precipitated by acids-
24 Practical Physiology.
Filter the solution through an ash-free paper and incinerate
the nuclein and paper together in a porcelain crucible, first
adding a few pinches of pure sodium carbonate. Cool, dissolve
the ash in weak nitric acid and test for phosphates (3) (4).
Besides nucleo-proteins, dead cells contain albumins and
globulins (though probably these are combined with nuclein
during life), inorganic salts, and two substances, lecithin and
cholesterin, which are constantly present in protoplasm, besides
traces of metabolic products and food materials.
Lecithins are complex fats which on hydrolysis yield gly-
cerine (1 moL), fatty acid (2 mol.), phosphoric acid (1 mol.), and
a poisonous basic substance, choline.
Note the physical appearance of a lecithin,' its solubility in
chloroform and ether, and the presence of phosphorus (demon-
strated).
Cholesterin is a crystallisable substance of large molecule
(Cj7H4gO) occurring in bile, medullary sheath of nerves, etc.
It is soluble in ether and in hot alcohol. The crystals are
rhombic plates, frequently having one of the acute angles de-
fective.
(75) Examine these dry, and then allow some 1 in 5 sulphuric
acid to run in under the coverglass — the crystals become red
and then violet. If a little iodine solution be added to the drop
of H2SO4 before applpng it to the crystals, a play of colours
results — violet, blue, green, and red.
(76) SalkowsKs Reaction. — To a chloroform solution of
cholesterin in a dry test tube add an equal volume of concen-
trated sulphuric acid. The upper (dhloroform) layer becomes
red while the lower layer shows a green fluorescence. On pouring
ofi the chloroform layer into a shallow basin the colour changes
to blue, green, and finally yellow.
Practical Physiology. 25
(77) Evaporate gently on open flame some of the choles-
terih solution on a piece of porcelain after adding a few drops of
nitric acid. A yellow residue remains which becomes red on the
addition, before cooling, of a drop of ammonia. This reaction
resembles the mure.xide test for uric acid. (See 261).
(B) CHEMISTRY OF EPITHELIAL TISSUES.
Besides the constitutents common to all cells, the epithel-
ial tissues contain Keratin and Mucin.
(78) Keratin. — A sclero-protein or albuminoid is the chief
constituent of the epidermic scales, hair, nail, hoof, feathers, etc.
With some horn shavings verify the following : —
The Keratin is insoluble in water and acids ; it swells in
cold alkali and partially dissolves in hot ; it gives the test for
"loosely combined sulphur" very distinctly (35), (36): also
Millon's and the xanthoproteic reactions for protein (the par-
ticles take on the characteristic colour) (1-5) (16).
(79) Mucin. — A conjugated protein of the gluco-protein
class, produced by "goblet" cells, and present in saliva and
mucous secretions generally.
Filter some saliva, add a drop of acetic acid — note the white
precipitate of mucin insoluble in excess of the acid. If a sufficient
amount of this precipitate can be obtained, boil it with weak
HCl for some time — cool, neutralise, and test for reducing sugar.
(C) CHEMISTRY OF CONNECTIVE TISSUES.
The Mucin of connective tissue differs in some particulars
from that of saliva. It can be extracted from tendons by lime
water or weak alkali, which destroy salivary mucin.
(80) Precipitate the mucin from a lime water extract of
tendon with acetic acid — collect^ the precipitate and hydrolise as
26 Practical Physiology.
above. Acid albumin will be present, formed from the protein
part and a reducing sugar from the prosthetic part of the mucin.
Collagen. — A sclero-protein, is a very insoluble substance.
(81) It gives the protein colour reactions Q.5), (16), and (17) ;
it swells up and becomes clear on being soaked in acetic acid ;
and on boiling with water it forms gelatine.
(82) Gelatine. — The hydrate of collagen ; in strong solutions
sets to a jelly — under i% it remains fluid at room temperature.
On a gelatine solution perform the usual protein reactions, and
note the peculiarities. (17) Biuret — violet colour ; (16) Millon's,
faint or absent ; (15) Xantho-proteic weak, usually only a faint
yellow appearing after addition of the ammonia ; (]8)-(20) gly-
oxyhc, Liebermann's and Molisch's tests — negative ; (conclusion
— the aromatic radicle only feebly represented in the gelatine
molecule) ; (21) boihng does not coagulate gelatine ; (22) nitric
acid causes no precipitate ; (23) Potassium ferrocyanide and
acetic acid cause a precipitate in weak solutions ; it is preci-
pitated by full saturation with ammonium sulphate and magnes-
ium sulphate, (25) and (28).
Elastln. — A sclero-protein from elastic tissue is insoluble
in water — it gives the protein colour reactions (15), (16). etc.
Bone and Dentine consist of two-thirds inorganic and
one-third organic matter.
(83) Take a piece of dried bone about the size of a large pea
and incinerate it in a porcelain crucible arranged as shown in
Fig. 1. Note the blackening which occurs indicating the pre-
sence of carbonaceous matter. Continue the incineration tiU
this completely disappears, cool, add about half full of the cru-
cible of hot distilled water and drop in nitric acid gradually till
no more of the residue dissolves. Filter to remove unconsumed
carbon, and test the filtrate for phosphates (3), calcium (9),
magnesium (10). Note the great preponderance of the first two
of these.
(84) Decalcify some of the same bone in diluted nitric acid
or in sulphurous acid and examine the residue for collagen (81).
(The chemistry of muscle is considered at the end of Chapter
III.).
CHAPTER III.
MUSCLE AND THE ACTION OF STIMULI
IN GENERAL.
(Examine slides showing the structure of striped, cardiac,
and non-striped muscle).
The physical properties of muscle which require special
study are EXTENSIBILITY and ELASTICITY.
(85) Prepare a Recording Drum for taldng a tracing by
covering it with a special glazed paper. First place the paper on
the desk with the smooth surface downwards and the gummed
edge at the far end facing you. Remove the cylinder from the
apparatus and place it across the paper with the upper end to
the right hand side ; wrap the paper firmly round the cylinder
and secure it by overlapping and fixing the gummed edge. Light
a piece of camphor placed on a porcelain slab and rapidly rotate
the paper in the flame or use a fish-tail gas burner applied to the
drum revolving in situ. Aim at getting a slight uniform coating
of soot all over the paper. If the first attempt fails, wipe off the
whole of the blackening and repeat the process. Next, prepare
the myograph lever for this experiment by reversing the lever
in the elbow-piece so that it can fall clear of the wooden part of
the myograph (Fig. 2) ; also, see that the scale pan and series of
weights are in readiness.
28 Practical Physiology.
(86) Pith a Frog by holding it in a cloth and inserting a stout
pin into the spinal canal at the junction of the skull with the
vertebral column. The correct point to insert the pin is in a line
with the posterior margin of the tympanic membrane of each
side. Move the pin quickly from side to side so as to separate
the brain from the spinal cord, then without withdrawing the
pin push it forwards into the cranial cavity and move it about
so as to destroy the brain completely, and then push it down
into the spinal canal and work it backwards and forwards so as
to destroy the spinal cord. If properly pithed, the limbs will be
perfectly limp and no reflex movements can be elicited on pinch-
ing the skin.
(87) Now dissect out the muscle — in this case the sartorius.
A good routine method in all muscle work is to prepare the lower
limbs and pelvis as follows : — Hold the frog by the hind limbs
(in a cloth to prevent heating the parts with the hand). Keep
the dorsal aspect uppermost and the head and spinal column
will droop downwards so that the upper ends of the iliac bones
form two small projections, thus marking the upper limits of the
pelvis. Cut through the vertebral column with scissors about half
an inch above the prominences and continue the incision through
skin and abdominal wall obliquely towards the pubis on each
side. Allow the viscera including the kidneys to fall forwards
and cut the rectum across close to its lower end. You have now
the hind limbs aud pelvis with the lower dorsal vertebrae.
Remove the skin by pulUng it right off from above downwards.
(88) The Sartorius Muscle will be found on the front of the
thigh arising from the symphysis pubis and ending on the tibia
by a short tendon. Pass a thread under this tendon, tie tightly,
making sure first that it is round the tendon and not round the
muscle which is easily divided by the tightening of the ligature.
Cut the tendon from the tibia making the incision as far away
from the ligatured point as possible, and then dissect the muscle
up to its origin. Repeat the same dissection on the other side
and then remove the whole front of the pelvis by two cuts with
with the scissors through the acetabula. The symphysis can
then be split into two, and so you obtain two preparations, each
consisting of sartorius muscle, its tendon with thread attached,
Practtcal Physiology.
29
Fig. 2.
Arrangement of the apparatus for the experiment
on the extensibility of muscle.
30 Practical Physiology.
and its bony origin. Pass a pin through, this bony part down
into the cork of the myograph and tie the thread to the upper
end of the short arm of the crank lever. Suspend the scale pan
from the lever at a point near its axis and adjust the level of the
lever so that it is horizontal or even points slightly upwards :
this may be done by sliding to or fro the elbow-shaped piece
which carries the lever and clamping it at the proper place. Next
bring the lever point against the blackened surface.
(89) Note that the vertical rod of the myograph moves as
a whole on its own axis, and that the movement is limited by
the "lever stop," a short arm of brass at the foot of the verti-
cal rod, which comes in contact with a brass guard. Before
applying the lever of the myograph to the blackened surface see
that this stop is close up against that side of the guard which is
next to the drum (A in Fig. 3). The writing point of the
lever can now be removed from its contact with the paper
without moving the foot of the stand (B in Fig. 3), and it can
be replaced with exactly the same degree of pressure against the
writing surface and therefore with the same position of the
point. Practice the use of this arrangement and note the use of
the T-piece to which the platform of the myograph is clamped
in correcting any tendency of the lever to leave the paper, or
to press too much against it as it rises or falls. Keep the
wooden part of the myograph as close up to the vertical rod as
practicable since the apparatus is more stable in that position.
The writing lever, the T-piece, and the lever stop should all
lie in the same plane or in parallel planes.
(90) Experiment on Extensibility.— Use a stationary drum,
i.e., disconnect it from the shafting and arrange it so that it can
be rotated by hand for a short distance at a time, and see that
it remains motionless at any point to which it has been rotated.
Apply the writing point to the blackened surface near the
top of the paper, using the lever stop as just described. Eotate
the drum gently so as to produce a fine horizontal base-line or
abscissa about four inches long, and then reverse the drum so
that the writing point is at the left hand end of this line.
Practical Physiology.
31
(^
Fig. 3.
The use of the lever stop.
32 Pbactical Physiology.
Place one of the weights gently on the scale pan and allow
it to extend the muscle and so trace a vertical line. After a
definite short interval of time rotate the drum so that a short
horizontal line is traced ; this must be of a definite length, say
three-quarters of an inch ; then place a second weight over the
first and allow it to act for the same time as the first, and then
rotate the drum again for three-quarters of an inch. Proceed in
this way to produce a stair-like tracing till no further extension
occurs. The line joining the bases of the vertical extensions
(ordinates) is the curve of extensibility.
(91) It is convenient to take at the same time the curve of
elasticity (or recoil). To do this, simply reverse the process,
taking off one weight after the other : allow equal time intervals
between each removal, and rotate the drum for an equal distance
each time.
(92) To preserve the tracing. — Eemove the cylinder from
the apparatus. Cut the paper along the seam and remove it
without smudging the tracing ; place it flat on the desk and write
neatly on it^the object of the experiment {e.g. extensibility
and elasticity of muscle) — the preparation used {e.g. frog's sar-
torius) — the date — your name, and any other markings that may
be necessary, e.g., in this case put in the curves of extensibility
and elasticity as dotted lines joining the lower ends of the vertical
lines. Now varnish it — hold the paper by its ends with black-
ened surface uppermost, allow the centre to dip into the varnish
and move it to and fro till the whole surface has been covered.
Pin it up by one end to drain off excess of varnish, and let it dry
for several hours.
ACTION OF ARTIFICIAL STIMULI IN GENERAL.
All living tissue possess excitability, that is, they respond in
some definite way to a stimulus ; in the case of muscular tissue,
the effect of applying a stimulus to the nerve is a visible short-
ening of the organ — so that a nerve-muscle preparation is con-
venient for the study of stimuli in general.
Practical Physiology. 33
A stimulus may be described as a change of condition or
environment of a tissue, suddenly produced and generally con-
sisting in the application of some form of energy, e.g., mechanical,
thermal, chemical, photic, electrical. They imitate the effects
of natural stimuli, e.g., the result of stimulating with electricity
the nerve supplying an organ is activity (or inhibition of activity)
of the organ ; but in some cases the result of artificial stimu-
lation is not quite the same as that of the natural mode.
The effect of a stimulus depends on (a) the strength of the
stimulus, and (6) the excitability of the tissue, so that a series
of results varying in intensity may be obtained, although there
are some exceptions to this rule, e.g.. cardiac muscle.
(93) Make a Gastrocnemius-sciatie preparation from a frog
in the following way. After pithing the frog (86), divide the
vertebral column above the pelvis and remove the skin from the
hind limbs as already described (87). Tie a short length of thread
round the tendon of the gastrocnemius muscle of one side and
divide the tendon beyond the ligature. Separate the muscle
from the leg bones up to the knee and cut the leg off below that
point. Expose and separate carefully the sciatic nerve where it
lies among the muscles on the back of the thigh, taking care not
to grasp the nerve with forceps or to damage it in any way. Then
turn the frog, place one blade of the scissors from above down-
wards deeply into the cavity of the pelvis and cut through the
symphysis pubis, and divide the frog into two by cutting up along
one side of the urostyle as far as the end of the vertebral column
which is also to be divided into two equal parts. Grasp one of
these with forceps and proceed to dissect out the lumbar plexus
and sciatic nerve of the side on which the muscle is already
prepared. Dissect out the whole length of the nerve down to
the knee joint ; lay the nerve over on the gastrocnemius and cut
through the femur and thigh muscles above where the nerve is
bent over The result is that you have a nerve-muscle prepar-
ation consisting of the lumbar plexus and sciatic nerve with a
piece of vertebral column to act as a handle to the
nerve, the gastrocnemius muscle with its bony attachments at
the knee, and the tendo Achillis with thread for fixing to the
writing lever.
34
Pbactical Physiology.
(94) Now prepare the myograph stand. — Cover the cork
platform with a piece of blotting paper moistened throughout
with normal saline. This should be exactly the same size as the
platform. Eeplace the crank lever in its usual position (Fig. 4),
if it has just been used for the extensibility experiment and tie
the thread to the short arm of .the lever — the best kind of knot
to use here is a loop knot so arranged that the thread can be
tightened up afterwards if necessary. There are usually two
holes on the short arm of the lever, and the one selected will
depend on circumstances : — the lower will give the larger curve
but gives more work to the muscle during contraction.
Fig. 4.
The upper end of the muscle is now to be fixed by pinning
the knee joint to the cork platform of the myograph stand : the
position of the pin will depend on the length of thread from tendon
to lever. When properly arranged the thread should be moder-
ately tautj the lever should be horizontal or should point slightly
downwards, and the head of the small screw on the elbow piece
which carries the lever should be raised so that, while it does not
support the lever, it prevents it from descending. These pre-
liminary manipulations (93 and 94) are required in practically,
all experiments on nerve-muscle work, and should be carried out
Practical Physiology. 35
as rapidly as possible, care being taken not to allow the tissue
to become warmed by contact with the hands, or by breathing
on to the preparation. Various methods of stimulation may now
be applied to the preparation, e.q., electric, mechanical, thermal,
chemical. The most universally employed of all these is the
electric method because of the ease with which its strength can
be graduated and the accuracy with which it can be applied to
one definite spot. As the other methods are destructive to the
tissue they may best be done at the end of some lesson after the
preparation has been used for other experiments. For con-
venience, however, they are described at this stage.
(95) Mechanical Stimulation is brought about by clipping
through the nerve with blunt scissors, pinching or tapping it.
It may be carried out more sj'stematically by an arrangement
whereby drops of mercury fall from a given height on to the
nerve.
(96) Thermal Stimulation. — Apply a hot wire to the cut end
of the nerve.
(97) Chemical Stimulation. — Apply crystals of sodium chlor-
ide in the same way.
(98) Experiment to show Stimulation with various strengths
of Galvanic Electricity. — Set up a Daniell cell as follows —
Rinse out one of the porous inner cell with water and almost
fill it with weak sulphuric acid ; place the zinc plate in this and
set the whole in the glass jar containing the copper and
copper sulpate solution. Attach wires to the zinc and copper
elements ; bring one to a mercury key and the other to the bind-
ing screw at one end of the wire of a monocord ; connect the
mercury key to the other end of the monocord wire ; attach
one wire of a pair of pin electrodes, also to one end of the monocord
and the other limb of the electrodes to the movable iron rider ;
(Fig. 5). Then push the pin electrodes slantwise into the myo-
graph cork near the broad end and lay the nerve across the pins : —
the nerve should lie on the moist blotting paper and should if
necessary be moistened from time to time with salt solution. It
will be noticed that one end of the monocord wire ( (a) in Fig. 5)
has two other wires connected with it, one from the soufce
86
Practical Physiology.
of the electricity, and an electrode wire. Place the rider
on the mnnocord wire near this end of it and close and
open the key : — Usually no result foUows because only a
small fraction of the available current will pass by the
electrodes in that position of the rider. Place the rider
further along the monocord wire and again close and open
the key • — If the muscle does not contract, move the rider
Fig. 5. (Exp. 98).
Practical Physiology. 37
still further along till contraction is obtained. Note that the
contraction occurs on closing and not on opening the key. With
full strength of the current, however, (rider at (6), (Fig. 5) con-
traction may occur on both closing and opening the key ; during
the flow of the current there is no contraction, at least not with
weak currents.
This shows how a stimulus may be graduated in strength ;
that the result may vary with the strength ; and that the stimu-
lation occurs during the change of condition and not during its
maintenance.
This last statement is more i'uily illustrated by the next
experiment in which the galvanic current can be sent into the
nerve almost instantaneously, or can be set up so slowly and
gradually that no stimulation results.
(99) For this purpose the Rheonome (Fig. 6) is used. It con-
sists of a base-board with a circular trough to hold a conducting
fluid such as saturated solution of zinc sulphate ; into this there
dip two strips of metal with binding screws to carry the wires
coming from the cell, a simple key being interposed. Two
metallic arms, which can be rotated on a vertical axle set in the
centre of the apparatus, also dip into the zinc sulphate
solution, and have the electrodes attached one to each ; the
nerve is laid across the electrodes as in the foregoing experiment.
When the arms are placed in line with the connections to
the cell ( AA ' in Fig. 6) a considerable amount of the electric current
will pass to and through the nerve because the electrodes are
now in communication with points which have a wide difference
in electric potential ; but when the arms are rotated so that they
lie at right angles to the line joining the battery communication
( B B ' ) no current passes through the electrodes because
they are now in communication with points of equal potential
(compare to Wheatstone's bridge, in position BBl all four
arms are equal). If now the movable arms are rotated
suddenly from position BB' to position AA' the current is
as suddenly sent through the nerve and contraction results, but if
the same movement be done slowly and steadily there is no
contraction.
38
Peacj'ical Physiology.
Fig. 6. (Exp. 99),
The Rheonome (von Fleischl's).
(100) Galvanic Stimulation of Sensory Nerves. -Clean the
electrodes, apply them to the tip of the tongue and send the
current through. A warm prickling sensation is felt during the
whole tinip of the flow and occurs whether the stimulus is slowly
applied or not.
(103) Experiment to show Stimulation with various strengths
of Faradic Electricity. — The form of induction coil used in prac-
tical physiology is generally known as the sledge inductorium
(du Bois Raymond) because the strength of the shock is gradu-
Practical Physiology. 39
ated by sliding the secondary closer to or further away from the
primary coil. In some forms (e.g., Porter's) this movement is
combined with and supplemented by a rotatory movement of
the secondary coil which has the same effect. The principle
of the apparatus is, that when a galvanic current is set up or
broJien, increased or diminished in amount in the wire of the
primary coil, it induces a "shock" or current of short dura-
tion in the closed circuit of the secondary coil, and the strength
of the shock depends on the proximity of the coils and the degree
of parallelism between the turns of wire in them.
If a nerve or excitable tissue forms part of this secondary
circuit, it is stimulated by the passage of the electricity through
it.
Connect a Daniell cell through a simple mercury key to the
terminals of the primary coil. Fit up the secondary circuit as
follows: — Attach a wire to each terminal of the secondary coil ;
fix the other ends of these to each side of a short-circuiting key,
and fix the ends of the pin electrodes to the same key, also one
to each side (Fig.7). Apply the electrodes to the tongue which
will thus complete the circuit. Open the short-circuit key,
place the secondary coil about 12 cm. from the primary, close
and open the mercury key — a sharp twinge or shock will be felt
on opening, possibly a lesser shock or nothing on closing the key.
Repeat this with the short-circuit key closed, and note the diff-
erence. Trace out the two separate circuits in this stimulating
arrangement. The primary circuit consists of the wire from one
pole of the cell to the mercury key, another wire from there to
the one terminal of the primary coil, the wire of the coil itself,
its other terminal, and the wire thence to the other pole of the
cell. The secondary circuit consists of the piece of tissue between
the pins of the electrodes (tongue in this case), one pin and its
attached wire, one side of the short-circuit key, a wire thence to
one teiniinal of the secondary coil, the long thin wire which forms
the secondary coU, its other terminal, a wire to the short-circuit
key, one side of that key, and the other electrode wire and pin.
Note how the closure of the short-circuit key diminishes the size
of this secondary circuit and, by providing an easier path for the
"shook," prevents the stimulus from reaching the tissue,
40 Practical Physiology.
These arrangements exemplify what must be present in
every case of stimulation by induced electricity, viz. — a com-
plete secondary circuit which involves the tissue to be stimu-
lated ; this must be in proximity to a complete primary circuit
through which a galvanic stream can be sent in an interrupted
or varying manner so as to produce shocks in the secondary.
Unipolar induction is an apparent exception in regard to
the completeness of the secondary circuit, see (104).
Note especially the means of varying or of interrupting the
galvanic stream ; in the above experiment it is a simple mercury
in other exneriments it will be found to consist of an automatic
key — (Neef's Hammer), or of a vibrating spring, or of the appar-
atus for obtaining a graphic record of muscle contraction — drum,
pendulum, or spring myograph.
Repeat this experiment with the secondary coil at different
distances from the primary and note how finely the stimulus
can be graduated.
(102) Now instead of the tongue (sensory nerves) apply the
electrodes to the sciatic nerve of a gastrocnemius- sciatic pre-
paration (93), and take a tracing of the effects of opening and
closing the key in the primary circuit. Begin with the coils so
far apart (say 35 cm.) that the stimulus is insufficient (" sub-
minimal"). Gradually approximate the secondary coil to
the primary till the smallest appreciable effect is obtained
("minimal" stimulus). Allow the muscle to record the height
of its contraction . on a stationary drum, having first taken a
base-line or abscissa. Write the distance of the coils from each
other under each attempt at stimulation. At first the closvire of
the mercury key gives no effect but some mark should be put
on the abscissa to indicate that it was tried. As the coils are
approximated the opening shocks cause larger contractions till
by and bye no further increase in height is obtained, the stimulus
is now said to "maximal," while those intermediate between
minimal and maximal are "submaximal." It will now be found
that the closing shocks begin, increase in efficiency and become
maximal in just the same way as the opening shocks but with the
coils closer together. During this part of the experiment the
Practical Physiology.
41
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42 Practical Physiology.
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opening shocks should not be allowed to reach the nerve other-
wise the preparation will rapidly become fatigued ; they must be
short-circuited by the key in the secondary circuit.
(103) Neef's Hammer (Fig. 8). — It is frequently necessary
to stimidate a nerve by a number of rapidly recurring shocks
and as this would be inconvenient and too slow if done by hand,
an automatic arrangement known as Neef's Hammer is commonly
used. Its mechanism is similar in principle to that used in
ringing an electric bell — that is, the galvanic current generated
on completing the circuit magnetises a piece of soft iron (D)
which then draws a spring away from contact with a platinum
point (C). This contact is an essential part of the circuit and
therefore the current ceases, the spring is no longer attracted by
the soft iron, it returns to its contact with the platinum point
and therefore the current is re-established. The number of
make and break shocks depends on the rate of vibration of the
spring which is usually about 30 per sec. If the secondary coil
is sufficiently near the primary there will be a stimulus at both
make and break, consequently doubling the above rate.
Connect the cell through a simple key with the two binding
screws on the middle and left hand pillars of Neef's Hammer
(BB^ Fig. 8). Adjust the height of the screw which carries the
platinum point till the sprang vibrates continuously. Connect
the secondary circuit through the short-circuit key to the pair
of electrodes and try the effect on the tongue and on the nerve
muscle preparation. Trace out the course of the current in the
primary circuit and note how the automatic 'make' and 'break'
is caused.
(104) Unipolar Induction. — Fit up Neef's Hammer as in
(103) and bring a single stout wire from one terminal of the
secondary coil to the nerve of the nerve muscle preparation
which for this experiment should be placed on a porcelain slab
or glass plate. Connect another wire to the gas piping or water
tap and bring its other end in contact with the muscle. Now
set Neef's Hammer agoing and in most cases the muscle will
contract. The reason is that some free static electricity is gen-
erated in the incomplete secondary circuit, and this, if discharged
into the earth through a living tissue, causes stimulation. It
Practical Physiology.
43
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44 Practical Physiology.
is therefore useless to employ a simple key in the secondary
circuit because inVsuch case there would still be one complete
wire from^the coil to the preparation at the end of which an
electric charge might accumulate and might find its way to
the earth through the muscle by means of the myograph stand,
etc. The short-circuit key, on the other hand, completely pro-
tects the preparation.
THE CONTRACTION OF MUSCLE.
So far. we have used the nerve-muscle preparation to illustrate
the action of stimuli in general. We shall now study more
closely the nature of the response to an artificial stimulus and
or this the nerve-muscle is the most suitable because the result
if a mechanical one which can be graphically recorded. When
as muscle or other organ is directly stimulated, the stimulus is
applied to the organ itself ; in indirect, stimulation the stimulus is
applied to the nerve supplying the muscle. (That muscle fibres
are themselves capable of responding to a stimulus is shown by
various facts including those of curara poisoning which may at
this stage be demonstrated).
(105) Graphic record of a muscle twitch or single
contraction. — This requires, in addition to the writing lever and
means of stimulation, some form of moving surface, such as a
revolving cyUnder, pendulum myograph, or spring myograph,
and in each case, if it is desired to find the latent period, the
the moving part must act as a key and cause stimulation at one
definite point in its motion.
Examine the "drums" used for muscle work in this labor-
atory. They are of two kinds — (1) de Burgh Birch's and (2)
Sherrington's. In the former, note how the motion is conveyed
from the grooved pulley to the revolving cylinder, how the brake
can be adjusted so that the cylinder is stopped immediately the
friction pulley leaves the under surface of the circular base- plate.
This pulley can be shifted nearer to or further from the central
axis, and so the speed can be varied ; the other means of varying
the speed is by using different combinations of pulleys on shafting
and on drum, always remembering that to increase the speed the
cord must come from a large pulley on the shafting to a small one
Peacttcal Physiology. 45
on the drum., and vice versa for diminished speeds. For this
experiment use such a speed as will cause the cyhnder to revolve
about once per second, or half that speed. Note the presence
of the two binding screws on the iron foot of the drum,
one of these is insulated ; note further that in its revolution
the pin which projects from the circular plate makes con-
tact with an insulated strip of brass which also has a
binding screw. Connect the insulated binding screw on the
foot of the drum with this brass strip by a wire ; it will
now be obvious that once in each revolution the whole
apparatus will act as a key, first closing and then opening the
primary circuit as the pin makes and breaks contact with the
strip of brass. Now make the following connections, zinc or
copper of cell to simple mercury key, thence to one terminal of
the primary coil ; copper or zinc of cell to one binding screw on
base plate of drum and from the other to the remaining simple
terminal of primary coil. Fit up the secondary circuit with a
short-circuit key and pair of electrodes. (Fig. 9). The mercury
key in the primary circuit is useful but not absolutely necessary.
Prepare a blackened cylinder (85) and a gastrocnemius-sciatic
preparation (93) as already described ; attach the thread
to the writing lever and adjust the level of the lever ; fix
the electrodes in the cork near the muscle and lay the nerve
across them. Find the correct position of the secondary coil
to give only a break shook : this may best be done by allowing
the pin to make contact with the brass strip so that the circuit
through the drum is closed, then open and close the mercury key,
with the short circuit key open. Bring the secondary coil as
close to the primary as is possible without producing a make
shock and then close the short-circuit key. Close the mercury
key and keep it closed for the rest of the experiment. Place the
writing lever against the blackened surface of the drum, with
the lever stop hard up against its guard (Fig. 3, A). Allow the
drum to revolve once to trace an abscissa, about a couple of inches
from the top of the paper. Remove the writing lever from the
paper, open the short-circuit key and allow the drum to revolve
once and give a stimulus. If the muscle contracts well, stop the
drum, replace the lever which should touch the abscissa already
taken, start the drum and after one revolution when it has gained
its uniform speed open the short-circuit key till just one twitch
46 Practical Physiology.
occurs, then close it immediately and stop the drum. Now mark
the point of stimulation ; to do this revolve the drum by hand
till the projecting pin is just about to leave or break contact
with the brass strip, raise the writing lever for half an inch or
less at this point on the abscissa. It is obvious that if the drum
had been moving with exceeding slowness the curve would have
begun and ended there, but owing to the rapid rate at which the
curve was taken the lever did not begin to rise till a short time
afterwards. The distance between the point of stimulation and
the beginning of the curve allows us to measure the period of
latent stimulation or latent feriod. To obtain the length of this
.r^gO.
^4^fff/f>//^y^,vr/^r^/7r/p/////////^,
Fig. 9. (Exp, 105).
A, B, the binding-screws on the drum, A is insulated
and connected by a wire to C, the brass strip with which
the pin D makes contact,
Practical Physiology. 47
and of the other periods of the curve it is necessary to find the
time value of the moving surface. This may be done by counting
the exact number of revolutions per minute, finding the time
occupied by one revolution and measuring the length of the paper
or the circumference of the cylinder — but a more convenient
way is to allow a tuning fork to write its vibrations beneath
the abscissa. Clamp the tuning fork on a myograph stand, arrange
it with the lever stop so that the style just touches the paper
when the stop is against the guard. Remove the fork by means
of the lever stop, nip, and suddenly release it, and return it to
its contact with the drum which should be revolving at its full
speed as when the muscle curve was being taken.
The latent period has already been discussed, the other
periods are those of contraction and of relaxation. To measure
these the writing lever should be raised and made to touch the
very summit of the curve, then allow it to drop and trace the
arc of a circle which will cut the abscissa at a point. This line
need not be traced all the way down, it is sufficient if it touches
the summit and the abscissa. Draw vertical lines downwards
from this point, from the point of stimulation, from the point
where the curve begins to leave the abscissa, and from where it
returns to it and estimate how much time is occupied by each
period. The tuning fork vibrations are 100 per second. Label
and varnish the tracing as in (92).
The influence of various conditions on the form of the muscle
curve may now be considered (106-111).
(106) Influence of Load. — Take a simple muscle curve as
above (105) with an unweighted lever which is supported by the
' 'screw-stop' ' when the muscle is at rest. Then load the muscle
by suspending the scale pan from the lever at a point as far from
the axis of the lever as is the attachment of the thread from the
muscle. In that way the muscle has no leverage and lifts the
actual weight employed (plus the weight of the lever itself). Add
one or more weights to the scale pan sufficient to affect the height
of the contraction, and take another curve over the first with the
same point of stimulation as before. ■ Repeat with further incre-
ments of weight till the muscle cannot hft the load, and find out
the actual weight lifted at each contraction including the weight
48 Practical Physiology.
which prevented contraction altogether ("absolute contractile
force"). It may be found that the first increase in weight causes
a higher contraction than in the un- weighted muscle, due to the
beneficial effect of tension on the excitability of muscle.
From the above experiment the v>orle done by the muscle can be
approximately calculated, if the actual amount of shorten-
ing in m.m is deduced from the 1 1 eight of the curve by
taking into account the magnification of the lever ; this
shortening multiplied by the weight lifted expressed in
grammes gives the number of gramme-millimetres of work.
The diminished rise of the lever is due to the contracted
muscle being simultaneously extended by the weight em-
ployed. An instructive experiment on the extensibility of
contracted muscle as compared to uncontracted may be
done as follows : —
(106a) Extmsihilily nf Contracted Muscle and the viork done by a
Contracting Uiiscle. — Dissect out the sartorius muscle of a
frog as described in (88) and arrange the myograph lever,
muscle preparation and scale pan as described in (90). Fit
up the induction coil to give an interrupted current (103)
and fix the electrodes in contact with the pelvic end of the
' muscle. Place the writing point against the drum, using the
lever stop as usual. Kotate the drum by hand so as to
produce a short length of abscissa. Begin at the left end of
this abscissa and record first the height of a contraction
when the muscle is weighted by the scale pan alone, using
an interrupted current, of a strength just sufficient to cause
maximal contraction, for say three seconds. Rotate the
drum about half an inch, add a weight to the scale pan,
record the resulting extension, and again stimulate and
record the height of the contraction. Move the drum again
for a similar distance, apply another weight, record the
extension and again stimulate. Continue these operations
till full extension is obtained, and be careful in each case to
move the drum the same distance, to allow the extending
weight to act for the same time, and to stimulate with the
same strength and for the same length of time.
A dotted line joining the bases of the ordinates gives the curve
of extensibility of muscle at rest, while a similar line
joining the tops of the ordinates gives the curve of
extensibility of contracted muscle, for the application of
the weight to the muscle before contraction may be assumed
to have the same effect as if the muscle were kept con-
tracted and then had the weight applied to it. l\ote that
the two curves approach each other, i.e., the extensibility of
contracted muscle is greater than that of muscle at rest,
and consequently it may be assumed that at a certain point
Pkaotical Physiology. 49
beyond where the curves cut, stimulation of the loaded
muscle would produce elongation (Weber's paradox). From
the same tracing the work clone hv the muscle may also be
calculated by estimating the magnification of the lever and
multiplying the actual shortening of the muscle by the
weight lifted as mentioned above.
One or other of the adductor muscles of the thigh may be
used in the above experiment instead of the sartorius.
(106b) After-load. — In experiment (106) the weights are
supported by the head of the screw-stop in the intervals
between contraction. If this screw be raised so as to
slacken the thread, the muscle contracts to an appreciable
extent before it begins to lift the weight ; this is termed
■" after-loading." ff the slackness of the thread is increased
it will be evident that the load will be applied to the
muscle at a later phase of the contraction. On a drum
revolved by hand allow the lever to trace a short abscissa
wiih the whole weight of the lever and scale pan with a
moderate load supported by the muscle, i.e., with the screw-
stop screwed far down. Stimulate with a single break
shock (102) and take the height of the contraction, the drum
being stationary. Now raise the screw stop so as to support
the lever and weight at a higher level and trace another short
abscissa ; repeat the stimulation. Raise the screw "still
further, trace yet another abscissa and again stimulate.
Continue this process till the contraction of the muscle no
longer lifts the lever. It will be found that in the later
contractions the weight is jerked up to a greater height
than in the initial contraction, and if the work done at each
contraction be estimated, it will be found to be greatest
with a certain small amount of after-load.
fl07) Influence of varying temperatures on the muscle
curve. — Take a simple muscle curve as above described (105)
at room temperature and mark the point of stimulation. Then
cool the muscle by placing a small block of ice in contact with it
for a few minutes and then take another curve with the same
point of stimulation. Remove the excess of water and raise the
temperature of the muscle by dropping on to it some normal
saline warmed to 30°C. in a test tube. Repeat the curve using the
same point of stimulation. Where two students are working
together it is convenient to insert two projecting pins into the
base-plate at points diametrically opposite so that two curves
are obtained at each, revolution of the drum.
Put on a tuning fork tracing and mark off the periods as in
(lOS). Note the advantage of the lever stop in this experiment.
50 Practical Physiology.
(108) Influence of repeated stimulations.— The same arrange-
ment may be used for this. Allow the muscle to cool to room
temperature or use a fresh preparation, and on a new abscissa
record successive contractions, allowing the drum to rotate con-
tinuously with the short-circuit key open ; or remove the writing
point from time to time and record only every tenth contraction
till complete fatigue sets in. The latter method shows the effects
more clearly, but in the former, the "staircase" effect may
sometimes be obtained, the first few stimulations causing
successively larger contractions. Two stimulating pins may be
used here also.
(109) Examine the arrangement of the ergostraph experiment
for the study of voluntary fatigue in human muscle.
(110) Influence of Veratrin on the Muscle Curve. — Make a
hyoglossus preparation by removing the whole lower jaw, and
the floor of the mouth from a frog. This should include the
tongue and hyoid bone. Tie a thread to the tip of the tongue,
attach the thread to the writing lever and fix the preparation
to the platform of the myograph by pushing the pin electrodes
through the floor of the mouth with one pin on each side of the
hyoglossus where it arises from the concavity of the hyoid plate.
Arrange the other parts of the apparatus as in (105) and find the
position of the secondary coil which will give a break shock.
Record a single contraction on a drum moving slowly — one revo-
lution in five to ten seconds. Remove the writing lever from the
paper, inject into the tongue a few minims of veratrin solution
and repeat the curve after waiting a minute or two. The
injection is to be made under the mucous membrane of the tongue
near the electrodes. The usual result is a very much prolonged
curve. With successive stimulation the e ffect wears off and then
returns on allowing the muscle to rest.
(111) Effects of a Second Stimulus applied soon after the
first, {a) Summation. — Arrange the rate of drum and apparatus
as for (105). Use a gastrocnemius sciatic preparation, and take
a simple muscle curve using a very weak stimulus so that the
contraction is minimal or sub-maximal. Insert a second screw
pin into the base-plate close to the first or fixed one, and repeat
the curve. The new curve will also be a simple curve but a
maximal one. Here the stimuli have been summated.
Practical Physiology.
51
(6) Superposition. — Move the secondary coil nearer to the
primary till a maximal break stimulus is obtained. Take a
simple curve on a new abscissa, using one pin only. Insert a
second pin in the same position as in the foregoing experiment,
i.e., close to the first one, and repeat the curve. Then move the
pins successively further and further apart, and so take curves
showing the effect of two stimuli separated by successively longer
intervals of time. The actual length of these intervals depends
on the rate of the drum. Mark on the abscissa the second point
of stimulation in each case. These curves may all be super-
imposed or they may be recorded on a new abscissa each time,
in which case a simple curve resulting from the first stimulus
should be put in for the sake of comparison. Take time tracing
as usual.
(112) Tetanus, or Compound Contraction. — In this experi-
ment a larger number of stimuli following each other at definite
intervals of time are applied to the nerve-muscle. Use the same
arrangement as above (105) but instead of the drum which in
these experiments acted as a key, insert in the primary circuit
a vibrating spring (Fig. 10). The spring has a point attached
Fig. 10. (Exp. 112).
which is made to dip into and out of the mercury cup of the simple
key. Arrange the rate of the drum to go as slowly as one revo-
lution in twenty to thirty seconds. Place the secondary coU
52 Practical Physiology.
so as to give only break shocks. Clamp the spring at the end
away from the contact-making point. Bring the writing lever
against the surface of the paper with the usual precautions (89).
See that the level of the spring is so adjusted that the contact-
making point is just clear of the surface of the mercury when at
rest. While the drum is revolving slowly depress the spring
with the finger, open the short-circuit key, and when the writing
point is at a suitable place on the abscissa, release the spring and
allow it to vibrate, and give about half a dozen stimuli, then close
the short-circuit key. Probably in this position of the spring,
where the vibration is at a slow rate, each stimulus will cause
a complete separate contraction. If two students are working
together, repeat this on another abscissa before changing the rate
of vibration of the spring. Then clamp the spring about an inch
to an inch-and-a-half nearer the contact-making end and repeat
the process. In this way a series of tracings may be obtained
showing all degrees of tetanus up to complete fusion. Avoid
fatiguing the preparation by not taking more than about six
contractions each time. If complete fusion of the contractions
cannot be obtained with the spring, owing to difficulty in approxi-
mating the stand of the spring to the mercury cup, put on a
tracing with the Neef's Hammer (103). Finally, take a time
tracing with the seconds clock and note how many stimuli per
second were sent into the nerve at each position of the clamp.
(113) Reaction of Muscle after Contraction. — Stimulate the
gastrocnemius muscle of one side till complete fatigue has set
in. Cut it across and express a drop of the muscle juice on to a
piece of glazed blue litmus paper. Place alongside it a similar
drop from the un-fatigued muscle of the other side. Allow both
to act for a few minutes ; wash off, and compare the acidity.
Usually the fatigued muscle is distinctly more acid.
Various other experiments on the contraction of muscle,
e.g., that on the rate of propagation of the wave of contraction,
on the nature of sustained voluntary contraction, on the muscle
sound, on the use of the dynamometer, etc., may be done by the
class or demon '(trated at this stage.
Practical Physiology. 53
THE CHEMISTRY OF THE MUSCLE.
Muscle consists of about 75% water, 20% proteins, 2% fat,
2% extractives, and 1% inorganic salts. Some ordinary butcher
meat has been freed from adherent fat, minced, and extracted
for twenty-four hours with 5% magnesium sulphate.
(114) Strain some of the extract through flannel till the
fluid is as clear as can be obtained. Determine the heat coagul-
ation temperatures of the proteins present (32). Since we are
dealing with muscle which has undergone rigor mortis, myosin
is the chief protein present. It coagulates about 56° C, but the
muscle and therefore the extract usually contains some para-
myosinogen which has not been converted into myosin, and a
coagulum due to this protein appears about 47° C. The
other antecedent of myosin, myosinogen, has the same heat
coagulation temperature as myosin so that the one cannot be
distinguished from the other by this method ; probably both
are present. Further coagula in small amounts may be found at
63° C. and 73° C, due to traces of serum globulin and serum
albumin respectively.
(115) Allow some of the MgSO^ extract to drop into distilled
water, a cloud forms due to the precipitation of the myosin. It
is also precipitated on dialysis and by full saturation with MgSO.^
and is therefore classified with the globulins.
(116) Dilute some of the MgS04 extract with three to five
times the volume of water, and keep it for some time on the water
batli at 40° C. A clot forms which is loosely coherent, and re-
sembles a precipitate more than a true clot. It may be due to a
formation of myosin from myosinogen and para-myosinogen
which have not previously formed myosin and which have been
extracted by the magnesium«sulphate solution.
(117) Boil some of the MgS04 extract, after diluting it with
water if there is much protein present. Filter, and test the
filtrate for protein by biuret or other colour test (15-17). There is
no result because all the proteins of muscle are coagulable by
heat. This filtrate contains the extractives and salts, and when
54 Practical Physiology.
concentrated by evaporation it resembles the common ' ' extract
of meat ' ' of commerce.
(118) A solution of " extract of meat " is put out. Test
it for protein (15-17) as above, and for carbohydrate (42'-43) ; only
traces of these, if any, will be found. Fat is also practically absent
except for lecithin, so that meat extract is of little value as a food,
although the presence of certain extractives, (nuclein bases,
creatinin, etc.) and salts make it extremely useful as a stimulant.
(119) Lactic Acid (sarcolactic). — Test the reaction of the
solution of meat extract with litmus paper. It is almost always
acid. Place some of the semi- solid extract in a dry test tube,
add some ether, and rub the extract and ether together against
the side of the tube with a glass rod. Allow it to extract for ten
minutes or longer ; then warm a porcelain basin containing a very
little water and pour the ethereal extract into it, keeping the eth?r
from the flame. The ether soon evaporates, leaving a watery
solution of lactic acid. Test this as follows : —
(120) Uffelmann's Test. — To a test tubeful of 5% carbolic
acid add one or two drops of dilute ferric chloride and mix. The
result is a fine blue solution known as Uffelmann's Reagent. Add
a few drops of the watery solution of lactic acid to the upper part
of the tube, the blue colour is replaced by a yellow.
(121) Phosphates. — Add some magnesia mixture and some
ammonia to a solution of meat extract. Ammonio-magnesium
phosphate is thrown down ; filter, and keep the filtrate for (122).
Wash the precipitate on the filter paper with one in three ammonia,
then run some dilute nitric acid through the paper and collect
the acid solution, add ammonia to this till a precipitate just
appears, again render acid with nitric acid, add amnion, molyb-
date and heat — a yellow precipitate of phospho-molybdate of
ammonia forms.
(122) Nuclein Bases. — To the phosphate-free filtrate as
obtained above add some silver nitrate solution, and if a whitish
precipitate forms (AgCl) add ammonia till it dissolves. A dark,
gelatinous precipitate forms of a silver-magnesium compound
Practical Physiology. 55
of hypoxanthin and xanthin. This may take some time to
appear (cf. 262).
(123) Remove the phosphates from another portion of meat
extract solution by adding some calcium chloride and ammonia ;
filter, and add a few drops of ferric chloride to the filtrate. A
dark precipitate of carniferrin forms, being a compound of iron
with phospho-carnic acid. Carnic acid has the composition of
anti-peptone.
(124) Creatin. — This is the most characteristic of the ex-
tractives of muscle. It can be recognised by the form of its
crystals (transparent rhombic plates). In acid solution it readily
passes over into its anhydride, creatinln, which is more easily
tested for. Some meat extract solution has been treated as
follows ■ — Basic lead acetate was added till no more precipitate
appeared, the fluid was then filtered, and the excess of lead re-
moved by passing HgS and filtering. The resulting solution was
then decolourised with animal charcoal and again filtered. By
this means the phosphates and many of the other constituents
are removed, and the solution can now be tested for the creatinin,
which has formed from the creatin owing to this and to the pre-
vious manipulation of the extract of meat.
(125) WeyVs Test for Creatinin. — Add a few drops of freshly
prepared dilute sodium nitro-prusside and then render the fluid
alkaline with NaOH or KOH — a deep red colour results which
becomes yellow on acidifying with acetic acid, and if the yellow
fluid is heated, it becomes green, and a blue deposit (Prussian
Blue) forms on standing.
(126) Jwffe's TaH for Creatinin. — To the solution add a few
drops of picric acid solution, and then make alkaline with KOH —
a deep reddish brown colour results, more marked on warming.
Glucose solutions give a similar result.
(127) If mince was used for the examination of the ash of
a tissue, see (I)-(IO) it will be unnecessary to consider further the
inorganic constituents of muscle.
CHAPTER IV.
NERVE TISSUE AND ELECTRO-PHYSIOLOGY.
Examine slides illustrative of the structure of nerve fibres
and their mode of termination, and of nerve cells stained by var-
ious methods.
That nerve fibres, in common with all other living
tissues, possess excitability and respond to a stimulus is shown
by the experiments already described under ' ' Action of Artificial
Stimuli in General," and those experiments on contraction of
muscle where the stimulation is indirect, i.e., applied to the nerve.
The response obtained on stimulating a nerve fibre is origination
and conduction of a nerve impulse which spreads as a wave along
the fibre, and which manifests its presence by the effect on the
end organ. Its presence can also be detected by the electric
change which accompanies it (142). That these two properties
excitability and condvctirity, are distinct from each other is shown
by various experiments in which one of these properties can be
affected while the other is not.
(128) Make a gastrocnemius-sciatic preparation (93) and fit
up the Neef s hammer stimulating arrangement (103). Connect
two pairs of electrodes to the terminals of the secondary coil
through a switch- commutator without crossed wires so that
the shock can be sent at will through either pair. A small
chamber has been made for the myograph stand in which the middle
portion of the nerve can be enclosed, and through which a stream
of CO2 can be passed (fig. 11.) One of the pairs of electrodes (6)
passes through the wall of the chamber so as to touch the
Practical Physiology.
57
Fig. 11. (Exp. 128).
enclosed part of the nerve ; tte other pair (a) is to be
placed on the nerve outside the chamber at the spinal end. In
this way one can stimulate the nerve outside or inside the chamber
before and after passing the gas. Make these arrangements and
find the position of the secondary coil which is just sufficient to
cause a slight tetanus of the muscle when the stimulus is applied
at (a) or at (6).
Now connect the chamber with the bottle containing the
marble chips, add HCl and allow the CO 2 to pass through the
chamber for several minutes ; then repeat the stimulation at
(a) and (6). If the experiment is successful, no contraction
follows the stimulation at (b), the inference being that the ex-
citability of the portion of nerve exposed to the gas has been
abolished while the conductivity has been unaffected. Vapour
of alcohol may be tried in the same way. It abolishes conducti-
vity while excitability is unaffected.
When a galvanic current of moderate strength is sent through
a nerve we have seen that contraction of the muscle follows on
closing the key and sometimes also on opening. When the
current is strong, contraction may continue during the flow, but
even where this does not occur there are are marked changes in
excitability and conductivity of the nerve trunk. These changes,
collectively spoken of as electrotonus will now be considered.
The positive and negative poles of the battery have almost
diametrically opposite effects on living tissues, and the influence
of the positive pole is known as anelectrotonus, that of the
negative as katelectrotonus.
58 Practical Physiology.
These efEects are well sliown on the beating heart of the frog.
(129) Examine the still beating heart of a pithed frog after
removing the sternum and pericardium. Note that during con-
traction (systole) the ventricle becomes pale and rigid, while
between the beats the muscle substance relaxes and becomes of
a dark red colour due to the contained blood. Connect a Daniell
cell to a simple key and use the bare ends of two ordinary wires
as electrodes. Place one of these ends, viz., that connected with
the zinc (negative pole or cathode) on some indifferent part of
the animal, e.q., under the liver or in the mouth, and place the
other (positive pole or anode) on the contracting ventricle and note
what occurs when the current is switched on. During contrac-
tion the muscle substance in the region of the anode remains
relaxed and a red flush can be seen around the spot in contact with
the wire. The result of application of the cathode in a similar
way is that during relaxation of the ventricle the area around
the wire remains pale and contracted. In each case removal of
the electrode causes momentarily the reverse effect.
The inference to be drawn from this is that the anode de-
presses the excitability of living tissue while the cathode increases
it, and that the same is the case with nerve may now be shown : —
(130) Influence of the positive and negative poles on the
excitability of nerve. — Arrange a nerve-muscle preparation on the
myograph and connect it to the writing lever in the usual way (9i).
Fit up (1) a simple stimulating arrangement (101), viz., one
Daniell cell and mercury key connected to the simple terminals
of the primary coil, and pin electrodes connected to the terminals
of the secondary coil through a short-circuit key (fig. 7) ; (2) a
battery to give a galvanic current of varying strength and direct-
tion (fig. 13), viz., connect two Daniell cells in series (zinc of one
to copper of the other) ; connect one pole of this battery through
a mercury key to one of the binding screws of the monocord,
the other pole directly to the other binding screw ; two ' ' lead-
ing-off ' ' wires are then taken from the monocord to the commu-
tator, one of these is to be fixed to one of the binding screws of
the monocord (A in fig. 13) — this binding screw will therefore
have two wires attached besides the wire of the monocord itself —
Practical Physiology
60 Practical Physiology.
the other leading-ofE wire is to be fixed to the movable rider :
the other ends of these leading-ofE wires are fixed one to each of
the lateral binding screws of the sliding commutator, or, if
Pohl's commutator is used, one to each of the binding screws
in line with the vulcanite top of the " cradle." The ends of
the electrode wires are now to be connected to the commutator.
If the Pohl's commutator is used, the cross wires are to be kept
in and the electrodes are connected, one opposite to each cross wire
and both at the same side. If the sliding commutator is used,
each electrode wire is fixed diagonally from corner to corner of the
sliding piece, and all four binding screws tightened on the wires.
Care must be taken that the wires do not touch where they cross.
Now prepare the non-polarisable electrodes ; saturate the
lower part of the boot with normal saline ; fill the small hollow on
the top of the ' ' foot ' ' with saline ; fix the electrodes to the glass
supporting rod and place them on the myograph stand so that
the nerve can be laid across the point of each boot as shown in
fig. 12. The electrodes should not touch the moist blotting paper
on the myograph platform. Last of all insert the amalgamed
zinc rods in the cavity of the ' 'leg' ' part and nearly fill that space
with saturated watery solution of zinc sulphate ; this must be
done very carefully with a pipette, and the zinc sulphate must
not come in contact with either muscle or nerve.
Now trace out the direction of the current from the positive
back to the negative pole of the battery. Note how the position
of the commutator changes its direction so that one or other
of the non-polarisable electrodes can be made the anode or cathode.
Examine also the way in which the monocord can be used
to obtain various strengths of current ; the two ends of the thin
monocord wire have been connected to the battery by ordinary
wires, and they therefore have a fixed difierence of potential with
a steady fall of potential all along the wire ; if the two leading-off
wires are widely separated in their contacts with the monocord,
that is, if the rider is placed close to the binding screw (b) (fig. 13)
which carries only one ordinary wire, the current is taken off from
two points at widely different potential and is therefore stronger
than if the rider is placed close to the binding screw (a) which
Peacttcal Physiology.
61
carries the other leading-off wire. For this experiment, begin
with the rider placed about the middle of the monocord wire,
and arrange the commutator so that the electrode nearest to
the muscle is the anode.
The object aimed at in the experiment, which can now be
proceeded with, is to stimulate the nerve with induced electricity
of a certain strength at a point which can be subjected at will to
the influence of the positive or of the negative pole.
^
Fig. 13.
Apparatus for investigating the influence of a galvanic
current on nerve.
62 Practical Physiology.
Arrange the upper part of the nerve on the non-polarisable
electrodes and place the ordinary pin electrodes from the induc-
tion coil on the nerve close to the lower of the two non-polarisable
electrodes ; with the key of the polarising (galvanic) circuit open,
find the position of the secondary coil which gives a minimal
stimulus on breaking the current in the primary circuit.
Record the height of this contraction on a stationary drum ;
rotate the drum to the left for about half an inch ; remove the
writing lever from the blackened surface by means of the lever
stop ; close the key of the polarising stream and thus subject
the stimulated part of the nerve to the influence of the anode ;
a contraction may follow on closing this key, but is to be disre-
garded ; while the polarising current is flowing replace the lever
against the drum and repeat the faradio stimulus used previously.
If the experiment is successful there will be no contraction or a
diminished height of contraction due to the anode having dim-
inished the excitability of the stimulated part of the nerve.
Eemove the writing lever again ; open the key of the polarising
current ; reverse the commutator ; again close the key of the
polarising current and again record the efiect of the break shock
from the induction coil. The stimulated point is now under the
influence of the cathode, the excitability is increased, and the
same stimulus is now more efficient and causes a higher contract-
tion. If the muscle remains in tetanic contraction during the
flow of the polarising current the strength of the current must
be diminished by moving the rider nearer to binding screw (o) ;
if the results are not obtained with the strengtTi of polarising
current at first employed, increase the current by moving the rider
nearer to (b), (Fig. 13).
(131) Influence of the galvanic stream on the conductivity
of nerve. — Use the same arrangements as in the foregoing ex-
periment, but shift the pin electrodes to the other side of the non-
polarisables, so that the nerve impulse must pass through the
portion of nerve affected by the galvanic stream before it cftn
reach the muscle. First test whether the impulse can pass when
the polarising current is not flowing. If so, close the key of the
polarising circuit and thus send a galvanic current along the
nerve (contraction may occur) ; now, while this current is on,
test whether the same faradic stimulus as was used before can
Practical Physiology. 63
cause contraction of tlie muscle ; usually it cannot. From the
results arrived at in the experiment on excitability (130) it will
be necessary to have the cathode next the stimulated point,
otherwise the result might be due to the effect on excitability.
The stimulated point should also be as far removed as practicable
from the non-polarisable electrodes. By special methods it can
be shown that the diminution of conductivity is due to the in-
fluence of the cathode.
(132) Experiment to show that with galvanic stimulation the
cathode stimulates on closing the circuit, the anode on opening,
and that the former is the stronger stimulus. Connect a Daniell
cell to the monocord through a mercury key as in (98), fig. 5,
using as electrodes two ordinary wires attached to the monocord
and its rider. Pith a frog, and prepare the lower half of the body
(87) ; lay it on the frog plate with the anterior aspect upwards ;
hook the bared ends of the electrode wire under the sciatic
plexus, one on each side, and secure the wires in position by
twisting each once round an ordinary pin and inserting the pin
in the cork plate. Beginning with a weak current, try the effect
of making and breaking the circuit. It will be found that con-
traction occurs at ' ' make ' ' in the limb affected by the wire
from the zinc (cathode) and at ' ' break ' ' by the anode, and the
former appears with a weaker current than the latter.
(With a certain strength of current both limbs contract at both
" make " and '' break." This is due to the formation of
secondary anodes and cathodes where the current passes
from nerve to the other tissues which form part of the
circuit).
The results in this experiment are explained by the fact that
any sudden (of. the rheonome, exp. 99) increase in excitability
acts as a stimulus ; thus, when the current is suddenly set up by
closing the mercury key the excitability of the tissue around the
cathode is as suddenly raised and stimulation ensues ; at the
same time the excitability around the anode is depressed
and remains so till the circuit is broken, when it suddenly
returns to or even rises above what it was before, hence stimula-
tion results here also, but it occurs only on opening the key and
is not so easily set up.
64 Practical Physiology.
(133) When a current of galvanic electricity is arranged to
pass through a piece of living nerve certain results have just been
seen to occur on opening and closing the circuit ; these results
vary according to the strength and direction of the stream, and
the whole series of results has been named ' ' Pflnger's Law of
Contraction." (Contraction, because it is usually performed on
the muscle-nerve preparation).
The arrangement of the apparatus in this experiment is
similar to that of (130) without the faradic stimulating part.
Make a nerve-muscle preparation and arrange it on the myograph
for recording the contractions ; fit up a polarising current of
two Daniell cells in series, a mercury key, commutator with crossed
wires, monocord, and non-polarisable electrodes, across which
the nerve is to be placed (fig. 13). Trace out the direction of the
current through the commutator ; when the anode is next the
muscle the current is said to be "ascending," if reversed, it is ' 'de-
scending." Begin with a weak current, i.e., place the monocord
rider so that the leading-off wires are close together. Close the
mercury key, keep it closed for several seconds, and then open it.
If no contraction follows on closing, increase the strength of the
current by moving the rider to a new position on the monocord
wire, till with a certain strength of current contraction occurs on
closing and not on opening the circuit ; record these effects,
reverse the commutator and repeat the closing and opening ;
mark on the tracing which direction of current was employed.
When contraction occurs on closing and not on opening the
effects are those of a " weak current." Increase the strength of
the current still more, till contractions occur at both closing and
opening with both ascending and descending currents ; these are
termed ' ' medium ' ' current effects. With still stronger currents
contraction may occur on closing a descending current and not
on opening, and on opening but not on closing an ascending
current ; these are termed ' ' strong ' ' current effects, and are
usually more difficult to obtain in ordinary class work.
Note that the terms " weak," " medium," and " strong "
do not refer to any definite strength of electric current (in voltage
or amperes) but to groups of effects obtained as one gradually
increases the strength of the current applied to a given nerve.
Note also that the effects may be to some extent mixed, e.g
Practical Physiology. 65
the ascending current may give ' ' weak ' ' effects when the
descending current of the same strength gives " medium," and
instead of no contraction one may obtain a diminished contrac-
tion.
If a very strong de cending current is used, i.e., strong
compared to the excitability of the nerve, the contraction on
closing the key may be a prolonged spasm (" closing tetanus "),
or this may appear on opening a strong ascending current.
(134) While a galvanic current continues to flow through a
nerve trunk it produces electrical disturbances in the neighbour-
hood of the electrodes which may be investigated by the galvano-
meter or capillary electrometer. These changes in electrical
potential may stimulate nerve fibres in the same trunk which are
not in contact with the electrodes and this accounts for what is
known as paradoxical contraction.
To demonstrate this, make a gastrocnemius- sciatic prepara-
tion ; in dissecting out the nerve note that it divides into two
branches ; follow the external (or peroneal) branch as far as
possible before cutting it across ; then finish the dissection in the
usual way. Lay the preparation on a glass slide with the
two branches of the sciatic clear of each other ; applv
a galvanic current to the peroneal branch by means of ordinary
pin electrodes connected through a simple key to a battery of two
Daniell cells — the gastrocnemius will contract.
(135) Electrical stimulation of nerves in situ. Apply faradic
and galvanic stimulation to the nerves of the arm. One electrode
is large and is placed on some indifferent part of the body such
as the abdomen or between the shoulders ; the other is small
so that it can be applied to those points where the motor nerves
come closest to the surface. Both electrodes are covered with
wash leather and are to be soaked in strong salt solution before
use. Note the response obtained in stimulating the various
nerves of the arm with faradic electricity. Galvanic stimulation
is carried out with the same electrodes. Use a commutator,
and make the small electrode first the cathode and then the
anode ; close and open the key and note the order in which con-
traction of the muscles supplied by the nerve appear as the
strength of the current is gradually increased. The order in
66 Practical Physiology.
which these contractions occur in the healthy subject are (1)
cathodal closing contraction, (2) and (3), anodal closing contract-
tion or anodal opening contraction ; (4r) cathodal opening con-
traction. In diseased conditions changes in the sequence of
these results occur, and are of importance in diagnosis, e.g., if
anodal closing contraction appears first it is known as the ' ' Re-
action of Degeneration."
(136) Resistance of nerve fibres to fatigue. That nerve
fibres cannot be fatigued easily is shown by stimulating a nerve
for a long time with repeated induction shocks, at the same time
preventing the impulses from reaching the muscle by lowering or
abolishing the conductivity of the nerve at some point between the
stimulating electrodes and the muscle. For this purpose an
ascending galvanic stream may be employed. After a long
period of stimulation, remove the "block" and it will be found
that the muscle responds.
(137) Double conduction in nerve fibres. Isolate the gracilis
muscle in the frog. It will be found on the inner and posterior
aspect of the thigh, its tendon of insertion being close to that of
the sartoiius. Dissect the muscle from below upwards ; note
its nerve of supply on the surface of the muscle next the femur ;
cut the nerve as far from the muscle as possible and then remove
the whole muscle, and lay it on a glass slide. It will be seen that
the nerve branches into two halves, one of which supplies the
upper and the other the lower half of the muscle. Cut the muscle
across so as to separate the parts supplied by these two branches.
Fit up an induction coil to give interrupted shocks. Stimulate
one of the branches of the nerve — both halves of the muscle con-
tract. The impulses which originated at the stimulated point
must have passed in both directions. It has been found that
each fibre in the main trunk of the nerve to the gracilis divides
into two where the trunk branches.
The above experiment (136) is apt to be confused with the
experiment to show Paradoxical Contraction (134).
(138) The velocity of tlie nerve impulse may be measured as
follows : — Make a nerve-muscle preparation in the usual way ;
fit up the apparatus for taking a simple muscle twitch but insert
Practical Physiology. 67
a switch-commutator without crossed wires in the secondary
circuit and attach two pairs of pin electrodes (cf. 128). Place
the electrodes on the nerve, one pair as near to the muscle as
possible and the other pair at the spinal end. Take two super-
imposed muscle curves on the same abscissa using a different pair
of electrodes each time. One muscle curve, viz., that obtained
with the ' ' spinal ' ' pair of electrodes, will rise from the abscissa
with a longer latent period than the other, and the space between
the points where the curves rise indicates the time taken for the
impulse to traverse the length of nerve between the two electrodes.
Put on a time tracing (tuning fork) and find the time value of this
interval: measure the length of nerve involved and calculate
the rate of transmission. The experiment is best performed with
the pendulum myograph.
The velocity of the impulse in human nerve in situ can also
be measured by stimulating the median nerve near the elbow and
at the clavicle, and taking a tracing of the contraction of the thumb
muscles with a Marey's tambour or transmission myograph.
For experiments on nerve cells see Chapter XI. (central
nervous system).
ELECTRO-PHYSIOLOGY.
(139) Galvani's Experiment. — Suspend the lower half of a
frog from an iron tripod by a copper hook passed under the
sciatic plexus of each side. Tilt the tripod so as to allow the leg
of the frog to touch the iron, a twitch will result. If unsuccessful,
see that the copper and iron make good electric contact, and hold
some iron or steel instrument, e.g., scissors, horizontally against
the I'.'g of the tripod, rub it to and fro so as to make good contfiot
and then allow it to touch the frog over the sciatic nerve. The
stimulation here is due to the electric current, produced by the
contact of dissimilar metals, flowing through the tissues.
(140) Galvani's " Contraction without Metals." Make a
nerve-muscle preparation and cut away the bit of vertebral column.
Place the muscle on a glass slide, raise the nerve by means of a bent
glass rod and allow it to fall on the gastrocnemius. Contraction
may occur, and is due to the differences in electrical potential of
the injured end of the nerve and the comparatively uninjured
muscle.
68 Peactical Physiology.
(Ml) Rheosoopie Preparation. Prepare two nerve-muscle
preparations. Arrange these on a glass slide so that the nerve
of the one lies on the muscle of the other. Tetanise the latter
through its nerve with an interrupted current. The muscle of
the second preparation also contracts. The term ' ' secondary
contraction " is applied to this result. It is due to the currents
of action in the first muscle and since the second nerve muscle
shows the presence of these currents it is termed the "rheoscopic
preparation."
(142) Demonstration of the electrical changes in dying and
in active tissue by the galvanometer and capillary electrometer.
CHAPTER V
THE BLOOD.
Histological Examination of Blood {See Schafer's "Essen-
tials of Histology"). Experiments on phagocytosis, diapedesis,
and amcBboid movement, also those on the efiect of water, salt
solutions, etc., on the corpuscles are now to be performed if not
already done.
(143) Enumeration of Red Corpuscles with the Thoma-
Zeiss or Thoma-Leitz apparatus.
Clean the special slide and coverglass so that they make
proper contact with each other ; this is shown by the appearance
of Newton's coloured rings reflected from the surfaces in con-
tact when viewed almost horizontally.
After cleaning the part, prick the skin so as to obtain a large
drop of blood without squeezing. Use the larger of the two
pipettes, viz., that marked 101 at the top of the bulb. Draw
blood up to 0.5 or to 1 and then diluting fluid (1 to 3% sodium
chloride) up to top of bulb, mark 101. Close the point and the
upper end of the pipette with the finger and thumb of one hand
and shake so as to mix thoroughly. Allow the diluting fluid in
the stem to run out, and then place a small drop of the mixed
blood and saline on the centre of the round glass platform of the
counting slide ; the drop should be of such a size that it almost
covers the platform when the coverglass is applied, and it must
not be so large that it overflows into the circular depression.
Apply the coverglass, lowering it by one edge immediately
70 Practical Physiology.
after placing the drop. Newton's rings should appear and re-
main permanent ; to bring them out a little pressure may be
brought to bear on the glass. Allow the corpuscles time to settle
(one to two minutes), and examine under the microscope. Focus
the lines which limit the squares. These squares are each equal
to T^o^li o^ ^ square millimetre and since the distance betweea the
surface of the round glass platform and the under surface of the
coverglass is one-tenth of a millimetre each square represents
^"0 oth of a cubic millimetre. The squares are further ruled into
sets of sixteen by lines which bisect each fifth row of squares
in both directions ; this is merely to facilitate the counting.
Count the number of corpuscles in a large number of squares,
say 50, and find the average in one square. In counting, include
the corpuscles lying on the lines only on two adjacent sides in
each, thus one may count as belonging to the one square, those
lying on the right hand line and the line next to the observer in
each case.
Multiply the average number per square by 4,000 and by the
dilution 200 or 100 according as to whether blood was drawn to
.5 or to 1. The result is the total number of red corpuscles in
one cubic millimetre of blood.
(144) Enumeration of White Corpuscles. — Prepare the
slide, coverglass, and sldn as before. Use the smaller pipette,
viz., that marked 11 at the top of the bulb. Draw blood up to
0.5 or to 1, and mixing fluid up to 11, so diluting the blood 1 in
20, or 1 in 10 respectively. The mixing fluid used in this case
must bfe one which will destroy the red corpuscles while it makes
the white cells more prominent — 1% acetic acid tinged with
gentian-violet is suitable. Mix the contents of the bulb, empty
the nozzle of diluting fluid and apply a drop of the mixture as
before. Count all the white cells in all the squares (^ 400)
moving the slide from side to side, twice to and fro. Multiply
the total number so obtained by 10 and by the dilution. The
result is the total number of white corpuscles in a cubic milli-
metre of blood.
(145) Differential count of number of varieties of Leucocytes—
Having estimated the total number of white cells per c.m.m.
Practical Physiology. 71
make and stain several large blood films and count the relative
numbers of neutro-philes, eosinophiles and lymphocytes present.
From this the numbers of the varieties can be calculated.
(146) Reaction. — Apply a drop of freshly drawn blood to
glazed litmus paper. Allow it to remain for about a minute
and then wash off with tap or distilled water. A blue spot in
the position occupied by the drop indicates the alkaline reaction
of the blood to litmus.
(147) Specific Gravity (Hammerschlag's Method). — The
method is based on the fact that when a drop of blood is placed
in a fluid of the same specific gravity as itself, it remains sus-
pended. A mixture of chloroform (Sp. Gr. 1.52) and benzol
(Sp. Or. 0.89) is made so that its specific gravity approximates that
of blood (1060). The mixture is put into a narrow jar or wide
test tube and a drop of blood is added to it from a fine pipette.
If the di'op sinks the mixture is of lower specific gravity than the
bl'.iod and some chloroform must be cautiously added, till the
drops remain suspended ; mix thoroughly after each addition
of chloroform. If the drop rises and floats on the surface, add
benzol in a similar way. When the mixture has been correctly
adjusted, take the specific gravity with an ordinary hydrometer
or urinometer.
(148) Lalcing of Blood. — To some defibrinated blood in
a test tube add once or twice its volume of water. It becomes dark
and at the same time transparent, so that print can be read
thrctugh a thin layer of it. If much water be added some cloud-
ing due to precipitation of the globulins in the serum may result.
To some blood in a test tube add a few drops of chloroform
or ether or solution of bile salts — laking occurs in each case.
(149) Crenation of Corpuscles. — To some blood add an equal
amount of strong solution of sodium chloride, — the mixture re-
flects more but transmits less light and therefore appears of a
brighter red than normal blood, but it is more opaque.
Coagulation of Blood. — This is due to the transformation of
the fibrinogen of the plasma into fibrin which forms a meshwork
of fine threads entangling the corpuscles. After a time the threads
72 Practical Physiology.
shrink and serum is expressed. The fibrin network has already
been seen (see Essentials of Histology).
Coagulation may be prevented by the addition of mag-
nesium sulphate, a soluble oxalate, or a soluble fluoride. .Tars
containing blood, to which these were added as the blood was
being shed, are put out and the following facts may be
demonstrated (]50)-(152).
(a) (150) Coagulation is not due to the Red Corjiuscles hut
to something in the plasma. Centrifugahse some of the MgSO^
blood so as to separate the red corpuscles out of the plasma or
pipette off some of the plasma if the corpuscles have settled in
the jar. Dilute some of the plasma so obtained with five to ten
times its volume of water ; divide into two portions ; to one add
a few drops of serum and place both on the water bath at 40° C.
A fine clear gelatinous coagulum will form in the one to which
the serum has been added, and possibly in the other also ; if
no clot forms in this second tube add some serum which has been
heated to boiling and then cooled. Replace it on the water
bath — no coagulation will occur. The inferences here are —
that MgS04. prevents coagulation only when in concentrated
solution, that the corpuscles do not form the clot proper (although
in ordinary circumstances they are entangled in the clot), and
that the property of the serum which causes coagulation is
destroyed by boiling.
(b) (151) Coagulation is -prevented hy the reinoval of Cal-
cium ions. — To some oxalated and therefore lime-free blood add
several drops (10 to 20) of 5% Calcium Chloride and place the
tube in the water bath at 40°C. Coagulation occurs. (Citrates
also prevent coagulation due to an action on the lime salts'and
may be similarly examined).
(c) (152) Coagulation is aided and hastened by the presence
of thrombo-hinase found in the watery extracts of all tissues. — To
some blood which has been mixed when shed with sodium fluo-
ride add, in one test tube a few drops of a strong solution of
calcium chloride, in another tube the same amount of the cal-
cium salt and also some watery extract of a tissue such as muscle
or liver. The second of these two tubes will show a clot much
sooner than the first when both are placed at 40°C.
Practical Physiology. 73
The" Composition of the Blood may be studied with oxal-
ated plasma freed from corpuscles by the centrifuge, or with
serum. The main bulk of the proteins consist of serum-albumin,
serum-globulin, and fibrinogen ; the last is absent from serum.
(153) Se-paration of the Proteins of Serum. — To some blood
serum add an equal bulk of saturated ammonium sulphate solu-
tion — a precipitate of serum globulin results. Filter and fully
saturate the filtrate with ammonium sulphate — the serum-
albumin is precipitated. Filter again and test the filtrate for
protein (25) (biuret test with strong NaOH) — no peptone present.
(154) Allow some serum to fall drop by drop into a large
amount of water slightly acidified with acetic acid or even into
plain water — a cloud results dvie to precipitation of some of the
globuhn. (See (29), the solvent power of the NaCl is lost on
dilution).
(155) Allow some serum to dialyse in a parchment tube
surrounded by water for two days — note the precipitate of glo-
bulin {eu-glohulin) — soluble in salt solution — and note also that
the filtrate from this precipitate gives another precipitate on
half-saturating with ammonium sulphate {pseudo-glohulin).
(166) Estimation of the amount of protein in serum. — Dilute
some serum to fifty times its original volume, using saline solu-
tion as the diluent. Fill an Esbach's Albuminimeter up to mark
U with this and add Esbach's Reagent up to mark R. This
reagent consists of a mixture of picric and citric acids. Let the
tube stand in a vertical position for twenty-four hours and then
read the height of the precipitate ; the figures on the tube refer
to grammes per htre. This method gives only approximate
results.
(157) Heat some diluted serum — the proteins are coagulated
— test the filtrate for proteins — (17) — no result, so that albumoses
and peptones are not present in demonstrable amount. If time
permits, find the heat coagulation temperatures of the serum
proteins (32).
74 Practical Physiology.
(158) After coaejulating the proteins of serum by heat as
above, the filtrate may be used to test for the presence of extracihes
such as glucose, urea, etc., and inorganic salts, such as chlorides,
phosphates, etc.
(1.59) The Guaiac Test is one frequently employed in testing
urine for blood, It is given by blood even when much diluted,
but a similar colour is given by various fluids such as milk,
saliva, etc., and it is therefore not an infallible guide in the exam-
ination of urine for the presence of blood. To some diluted blood
add a few drops of tincture of guaiac — the yellowish white guaiac
resin is precipitated on contact with the watery fluid. To this
mixture add some ethereal solution of hydrogen peroxide ("ozo-
nic ether"). A deep blue colour develops.
Haemoglobin. — Haemoglobin is the chief constitutent
of the red corpuscles. In some animals it is present as a less solu-
ble variety than in others and can therefore be made to crystallize
more readily (31).
(160) Iron is present in Haemoglobin. — To some dried
haemoglobin in a test tube add about double its bulk of 1 in 4
nitric acid, and heat for some time ; cool, filter and test the filtrate
for iron by adding ammonium sulphocyanide (red colour) or
freshly prepared potassium ferrocyanide (Prussian blue).
Solutions of haemoglobin, its compounds, and derivatives
give characteristic absorption spectra. These may be conveniently
examined with a small spectroscope — the prisms of which are
so arranged that the whole spectrum is visible at one time.
Examine the spectroscopes provided. Note that the width of
the slit can be adjusted according to light requirements, and that
the spectrum can be focussed by adjusting the eyepiece. Direct
the silt to the sky and arrange it so that Fraunhofer's lines are
visible, then direct it towards a sodium flame and note the posi-
tion of the D hne. It is usual to hold the spectroscope so that
the red end of the spectrum is on the observer's left hand side.
In all work on the spectra of pigments the degree of dilution of
the substance examined is of importance and it is generally
possible to add the diluting fluid in such a way that various
strengths of solution can be seen in the one tube.
Practical PHYSioiiCGY. 75
(161) Oxyhaemoglobin. — Take some defibrinated blood in
a test tube and run in some water directly from the tap, holding
the tube obliquely under the end of the pipe : allow the water
to continue running after the tube is full. In this way one obtains
a solution of the oxyhaemoglobin so diluted that the upper part of
the tube contains almost pure water, the lower patt blood, and
the middle part all gradations between the two. The corpuscles
are laked of course.
Adjust the spectroscope as described above, and hold it in
the right hand ; dry the outside of the test tube containing the
diluted blood and place its upper end against the slit, holding the
tube by the lower end with the left hand.
On looking through the spectroscope probably no bands will
be seen. Gradually raise the tube so as to bring a stronger solu-
tion of oxhaemoglobin in front of the slit. Two bands will appear,
one narrower than the other and nearer the red end of the spect-
rum. In stronger and stronger solution these two bands fuse
into one and broaden out so as to obscure the whole spectrum.
Using the strength which shows the two bands clearly direct
the spectroscope towards the sodium flame (the sodium is appUed
to an ordinary gas jet so that the whole spectrum is visible
with a bright line in the position of D.). It will be seen that both
bands lie between D and the violet end of the spectrum close to
D. The band next D is the narrower and is more sharply defined
than the other.
(162) Haemoglobin — (Reduced Haemoglobin) — Prepare a
solution of oxyhaemoglobin of such stren^h as to show the two
bands, then add a reducing agent — ammonium sulphide is gener-
ally employed. Keep the mixture at body temperature to
hasten reduction. The spectrum now shows a single broad band,
as if the interval between the two bands of oxyhaemoglobin had
been obscured. Examine the spectrum in various strengths of
solution using cold boiled water or weak ammonium sulphide
dilution to prevent re-oxidation (tap water contains dissolved
oxygen).
Stokes' solution is a better reducing agent than ammonium
sulphide. It consists of 2% ferrous sulphate in 2% tartaric acid
solution, and just before use, sufficient ammonia is added to
76 Practical Physiology.
make it slightly alkaline. When reduction is efiected by ammon-
ium sulphide, a narrow band may appear to the left of D, this
does not occur when Stokes' reagent is used.
(163) Carbonic oxide haemoglobin (HbCO). — CO unites
with haemoglobin in the same proportion as Og but forms a
very much firmer compound. Take some blood in a test tube ;
add water to liberate the haemoglobin and so facilitate the test ;
allow some coal gas to run into the top of the tube ; close the
open end of the tube with the finger and shake ; repeat the intro-
duction of gas and shaking several times. The blood has now
become very pink — easily seen in the froth. Examine with the
spectroscope ; diluting with water as in (161). Two bands will
be seen apparently just similar to those of HbOg although the
one next the violet end of the spectrum is slighty narrower than
the corresponding one of HbOo.
The real distinction between the two pigments is that HbOg
is easily reduced while HbCO is not. Add Stokes' Reagent or
ammonium sulphide and keep at 40°C. No reduction occurs
even after prolonged action.
Another good distinction between the two pigments is tha t
when an HbCO solution is much diluted with water it remains
pink while HbO^ when dilutpd shows a yellow colour. This
can be easily demonstrated by making a very dilute (yellow)
solution of HbOg ^vith blood and saturating a portion of it with
coal gas.
(164) Methaemoglobin. — To some diluted blood add a
few drops of potassium ferricyanide (other substances which may
be used are amyl nitrite, potassium chlorate, permanganates,
etc.). Keep at body temperature. This solution becomes brown
and the spectrum now shows in strong solution a band between
D and the red end of the spectrum. This is the characteristic
band ; others may be seen on dilution in the position of the two
bands of oxyhaemoglobin and a fourth band nearer to the violet
end of the spectrum. Add to the still warm solution a few drops
of ammonium sulphide and watch the changes that occur. First
the spectrum of oxyhaemoglobin appears and then that of re-
duced haemoglobin.
Practical Physiology. 77
(165) Acid Haematin. — To some diluted blood add some
acetic acid (about one-tenth its bulk of glacial acetic acid is suffi-
cient). Keep on the water bath at 40° for five to ten minutes
and then heat to nearly boiling. Too rapid heating precipitates
the proteins and causes turbidity. Slow heating obviates this
by allowing acid albumin to form. Examine the dark brown
solution of acid haematin, diluting if necessary with acetic
acid. A band in the red will be seen in much the same position
as the chief band of methaemoglobin. A few drops of ammonium
sulphide cause no change (compare to Met-Hb). Further
addition of the reagent causes a precipitate which dissolves in
KOH and the fluid may then show the spectrum of haemo-
chromogen (alkaline haematin reduced by the sulphide).
(166) Alkaline Haematin and Haemochromogen. — To diluted
blood add some KOH. Warm gently at first then heat to near
boiling, cool, and shake with air to restore oxygen to the com-
pound. A faint band of alkaline haematin will be seen to the
red side of D, but is frequently indistinct. On adding a reducing
agent, however, a very distinct spectrum appears, viz., that of
haemochromogen, or reduced haematin. This consists of two
bands between D and the violet end of the spectrum, but not
close to 15. This distinguishes it from HbCO and HbO.2, and,
further, the band next D is much stronger than the other, and
persists in weaker solutions, so that it may be the only one to
appear.
This formation of haemochromogen is a very delicate test
for blood.
(167) Haematoporphyrin. — Take about an inch depth of
pure clear sulphuric acid in a test tube and allow one drop of
defibrinated blood to fall into it. Spectroscopically it shows two
bands, one on either side of the D line — that to the red side of
it being the narrower of the two.
(168) Haemin is the hydrochloride of haematin. It forms
characteristic dark brown rhomboid al crystals on heating dried
blood with glacial acetic acid and a crystal of NaCl. (See
Essentials of Histology).
78 Practical Physiology.
(169) Quantitative Estimation of Haemoglobin.— In clinical
work this is done by diluting the blood to a certain extent in a
gradijated tube and comparing the tint of the solution with the
tint of a standard colour in a similar tube. The extent to which
it is necessary to dilute the examined blood before the colours
become equal in depth gives directly the percentage of haemo-
globin. In Haldane's modification of Gowers' apparatus the
standard consists of a 1% solution of blood saturated with CO
and preserved in a sealed tube. The estimation is made as
follows : — Blood is drawn up to the mark 20 cm.m. on the pipette
and blown out into a small amount of distilled water previously
placed in the dilution tube. A stream of coal gas from a narrow
glass tube connected to the gas tap is run into the dilution tube
so as to replace the air by gas containing CO. Close the end of
the dilution tube with the finger and invert several times to allow
the haemoglobin to become saturated with CO ; then add dis-
tilled water drop by drop till the tint of the diluted blood appears
to correspond exactly with that of the standard. In comparing
the two, they ought to be viewed by transmitted light from some
white surface such as bright clouds or the opal shade of a reading
lamp, and the tubes should be frequently transposed. Read the
percentage and then continue the dilution till the sample is just
appreciably lighter than the standard, read again and take the
average of the two figures. This represents percentage of
haemoglobin.
The capacity of the dilution tube up to the 100 mark is 2 c.c,
and since 20 cm.m. of blood is taken, dilution up to 100 gives
1% solution. The standard tube contains a 1% solution of blood
which had an ' ' oxygen capacity ' ' of 18.5%, which means that
each 100 c.c. of this blood contains an amount of haemoglobin
capable of carrying 18.5 c.c. of oxygen when fully saturated with
air (or O2). From this one can find the oxygen capacity of the
sample of blood taken. If, for example, the blood shows 108% as
compared with the standard, then its oxygen capacity is \%% X
18.5 = 20 c.c. Further, the relative amount of haemoglobin
per red corpuscle can be stated if a blood count is made at the
same time. Thus, if the haemoglobin is 50% while the corpuscles
number 80% of the usual, then the haemoglobin per corpuscle
is reduced to five-eighths of the normal. In some forms of anaemia
Practical Physiology. 79
the haemoglobin percentage may be below normal, but"] the
haemoglobin value per corpuscle may be increased.
There are various other methods used clinically where
the standard consists of a series of glass discs coloured to represent
various percentages of haemoglobin (Oliver's tintometer) or a
wedge of coloured glass similarly coloured (Fleischl's haemometer)
or a drop of blood on white filter paper is compared with standard
coloured papers (Tallquist's method).
The ' ' oxygen capacity ' ' of blood is an exact gauge of its
functional capacity as an oxygen carrier. This can be determined
by the blood gas pump, or by the ferricyanide method using
Haldane's apparatus. (Demonstration).
CHAPTER VL
CIRCULATION OF THE BLOOD.
(170) Study the gross anatomy of a mammalian heart (of
sheep or ox). Note the following : — The oblique groove on the
front of the heart filled with adipose tissue and extending from
the base nearly to apex, it indicates the position of the inter-
ventricular septum ; a similar groove on the posterior aspect of
the heart ; the aurioulo -ventricular groove at the base of the
ventricles ; the right auricle and its appendix ; the right
ventricle, thinner walled than the left ; the corresponding
parts on the left side ; the openings of the superior and
inferior cavae into the right auricle ; the openings of the
pulmonary veins into the left auricle ; the aorta and pulmonary
artery. Slit open all the cavities and note the following : — The
musculi pectinati in the appendices ; the Eustachian valve or fold
on inner surface of the right auricle ; the tricuspid valve in the right
auriculo-ventricular opening ; the chordae tendineae, musculi
papillares and ' ' moderator band ' ' of the right ventricle j the
pulmonary valves with their sinuses ; the interior of the left
auricle ; the mitral valve and the thick wall of the left ventricle ;
the aortic valves, their sinuses and the origin of the coronary
arteries.
Slides illustrating the structure of the heart and vessels
should now be examined.
(171) Action of the valves. — In another heart open the
auricles and clear away the tissue so as to expose the auriculo-
ventricular openings. Allow a fine stream of water from the tap
to fall into the centre of the opening of either side — the valve
Practical Physiology.
81
flaps will be seen to float up so that the upper surfaces of the edges
come into contact. Examine the semilunar valves in the
same way.
Make a small opening in the apex of the left ventricle, plug
it with one forefinger, fill the ventricle with water so as to float
up the valves and suddenly withdraw the finger — the valve flaps
will be seen to fall back against the sides of the ventricle.
(172) The beat of the frog's heart. Pith a frog. Make a
longitudinal incision through the skin from the floor of the mouth
to the pubis, and another transverse incision opposite the fore limbs.
Dissect back the flaps of skin. Incise the abdominal wall to
one side of the linea alba so as to avoid cutting the anterior
abdominal vein. Continue the incision upwards on each side of the
sternum, which can then be removed. Expose freely the beating
heart. Note the delicate pericardium, and then dissect it oS. The
frog's heart differs from that of mammals in that it consists of a
sinus venosus which receives the veins, a right and left auricle, one
ventricle, a bulbus arteriosus and two aortic arches. The parts
contract in the above named order, the two auricles contracting
simultaneously. Try to follow the order of the movements with
the eye as far as possible, then proceed to take a tracing (cardio-
gram). Arrange the elbow piece of the myograph stand at the
broad end of the cork platform ; fit it with a straight lever
instead of the crank lever (fig. 14) ; let the short arm of the lever
Fig. 14.
The crank myograph arranged for heart work.
82 Practical Physiology.
project over the heart of the frog ; insert a fine hooli into the
muscular tissue of the tip of the ventricle without grasping the
ventricle or damaging it in any way ; tie a short length of thread
to the hook ; make a running loop on the other end and pass it
over the end of the short arm of the lev#r ; adjust the parts so
that the heart is stretched vertically upwards, the thread to
the lever must be quite perpendicular ; try various positions of the
loop of thread along the lever till a point is found that gives a
maximum amou)it of movement of the writing point. Before
beginning to talre the tracing look for the pericardial ligament —
an extremely fine thread of tissue which connects the base of the
ventiicle to the pericardium — cut it through. Blacken a drum
paper and arrange the driving cord for a slow rate of drum, one
revolution in two minutes. Take a normal tracing of the beat,
using the lever stop as in muscle work. Record also the effects
of cooling the heart by applying ice to the sinus, and the effect
of warming the heart by pouring over it some saline heated to
30° C. Put on a time tracing with the seconds clock, write the
description on the paper, and varnish it as before.
(173) Listen with the simple wooden stethoscope, and the
binaural stethoscope to the heart sounds of a fellow student. The
first soimd is most distinct where the cardiac impulse or apex
beat is felt, the second sound is to be listened for at the second
right costal cartilage, but both sounds can be heard in each of these
two areas.
(174) The cardiac impulse or apex beat.— This is a movement
of the chest wall over the apical part of the heart which can be
seen and felt in the fifth intercostal space, a little to the inner
side of a vertical line through the middle of the clavicle (mammary
line). It is due chiefly to the hardening of the muscular fibres
and the increased convexity of the front of the ventricle which
occurs during systole. Palpate the area involved and take a
tracing of the movement with a cardiograph. This consists of two
tambours each covered on one surface with an indiarubber
membrane ; they are connected by rubber tubing ; the one
tambour is the recording one, and acts on a writing lever fixed
Practical Physiology. 83
in a stand ; the other, the receiving tambour, is fixed in a hollow
wooden shield so that when the edges of the shield are pressed
against the fifth and sixth ribs the button attached to the rubber
membrane comes in contact with the point where the movement is
greatest. Instead of the latter a small glass funnel may be pressed
over the part so as to make a small airtight chamber containing
the beating area, the rubber tubing being slipped over the stem
of the funnel.
The cardiograph should be held in position by elastic tapes
passed round the body, the lever of the recording tambour should
be horizontal, and the whole apparatus should be moderately
distended with air before taking the tracing. Use a moderate
rate of drum, about three revolutions per minute. The tracing
will probably shoAV large waves due to the respiratory movements
and much smaller projections due to the heart beats. Record
the time value with the seconds clock. The button of the receiv-
ing tambour must be very accurately placed ; if it is put in con-
tact with the chest wall to one side of the apex beat a curve will
be obtained which is almost the exact reverse of the normal,
because the chest wall is drawn in near the apex beat while it
protrudes at the centre of the area.
(175) The Pulse. — Feel the pulse in the radial artery at the
wrist by compressing it against the bone. Count the number of
beats per minute while the subject is standing, sitting and lying
horizontally.
Sphygmograms. — Examine the mechanism of a Marey or
a Dudgeon sphygmograph. In both there is a clockwork arrange-
ment to give a moving surface which may have its time value
determined by noting how many seconds it takes to run through
a certain length of paper (usually six inches in fifteen seconds).
The clockwork is set in motion by releasing a small lever on the
top (Dudgeon) or at the back (Marey) of the instrument. The
writing lever is acted upon by the pulse beat through a snjall
spring armed with a button which presses on the artery. The
amount of pressure exerted by the spring can be varied by means
of an eccentric. In Marey's sphygmograph the lever is a long
one and is acted upon directly by the spring. In Dudgeon there
is a system of levers and it has the advantage that the writing
84 Pbactical'^Physiology.
point moves at right angles to the direction of movement of the
paper.
Use of Dudgeon's Instrument.-Before applying the instrument
wind the clockwork ; see that the paper wiU run through easily
when placed between the moving roller and the two small guiding
wheels, and blacken one or two papers in the camphor flame.
Rest the subject's arm in an easy position on your knee and feel
for the position where the beat is most distinct ; the spot may
be marked with a skin pencil or with ink. Then apply the button
of the spring to the spot marked and secure the tape round the
back of the wrist. Put a blackened paper in position and allow
it to run in till it supports the writing point of the levers. Adjust
the tightness of the tape and the pressure of the spring on the
artery (by means of the eccentric) till a maximum amount of
movement is obtained ; release the clockwork and allow the paper
to run through to near the other end ; then stop the clockwork,
remove the paper, label, and varnish it.
Use of Marey's Instrument. — The general directions are
the same as above ; the arm should be bare to the elbow and
supported on the special splint which accompanies the instru-
ment ; the spring is applied to the pulse and the instrument
placed with the writing lever pointing towards the elbow, and
fixed by tapes passed round the arm. Adjust the pressure of
the spring on the artery by means of the eccentric till a maximal
amount of movement is obtained ; then adjust the level of the
lever, take the tracing, label, and varnish it.
(176) Blood Pressure. — (Demonstration). A rabbit is anfes-
thetised with paraldehyde ; its carotid artery exposed, and a
' ' wash out ' ' canula inserted ; the canula is connected to a
mercurial manometer or to a Hurthle's manometer, the connecting
tube, which should be inelastic, being filled with a fluid capable of
pjeventing coagulation, e.g., 2% sodium citrate. The movements
of the manometer are recorded on a revolving drum in the usual
way and the effects of injection of " pressor " substances such
as adrenalin or of " depressor ' ' substances recorded ; also the
effects of gravity on the pressure, of stimulation of the vagus,
etc.
Practical Physiology. 85
Venous pressure can be demonstrated in the same animal
by means of a water manometer.
(177) Capillary Pressure is estimated indirectly as follows :
(Von Kries' method). A small scale pan is suspended by threads
carried from each end of a small rectangular piece of glass. The
under surface of the glass has a piece of cover glass of known area
fixed to it with Canada balsam ; the whole is suspended from
the finger in such a way that the small piece of glass presses on
the delicate pink skin at the root of the nail, weights are then
added to the scale pan till the skin area under the small piece of
glass becomes pale. The pressure is calculated as follows : —
Suppose the area of the skin blanched = 4 sq. m.m., and the
weight of the apparatus plus the added weights amount to 1 grm.
(^1000 c.m.m. of water) ; then the height of the column of water
which would be required to overcome the lateral pressure in the
capillaries of the area considered would be -L-°^ = 250 m.m.,
or 18.5 m.m. of mercury. The results obtained by this method
are only approximately correct.
(178) Arterial Blood Pressure in Man. — The arterial blood
pressure can be determined with a fair degree of accuracy by
noting the constricting force necessary to compress the tissues
of a limb to such extent that the pulse is obliterated (systolic
pressure) and till maximal pulsation is obtained (diastolic
pressure).
The constriction is produced by fastening a broad leather
band round the limb, generally the upper arm. To the inner
surface of this belt an elongated elastic bag is fixed, and this
communicates with the pump and with a manometer which may
be in the form of a hollow spring acting on an indicator (Hill and
Barnard's sphygmometer), or a mercurial manometer. The bag
is distended by pumping air into it and the pulse at the wrist is
continuously observed. When the pulse can no longer be felt
the systolic pressure has been reached. The air is then gradually
released and the oscillations of the manometer noted ; when
these become maximal around a certain point on the scale, the
diastolic pressure is indicated by that point.
The results are stated to be from 6 to 10 m.m, above the
86 Pbactical Physiology.
true figures owing to the force required to compress other tissues
in the limb besides the artery.
The diihculties in applying the method are chiefly in deter-
mining the "end points," i.e., exactly when the pulse is com-
pletely lost and exactly at what stage the pulsation is maximal.
These are to a great extent overcome in the instrument devised
by Erlanger (in Howell's laboratory) ; in this apparatus a tracing
is taken of the pulse beats during the observation and the end
points are easily discerned.
The average pressures in the brachial artery are 110 m.m.,
systolic and 65 m.m. diastolic (Erlanger).
Various instruments have been devised for estimating blood
pressure in the radial artery at the wrist or other accessible
vessels by direct pressure over the vessel, but these are less
reliable than those which depend on constriction of the whole
limb.
(179) Velocity of the Blood Stream. — Demonstration of
the use of the stiomuhr in an artificial scheme of the circulation.
The instrument consists of two glass bulbs fixed in a base plate
which is double ; the inlet and outlet tubes are connected with
the central and peripheral ends of the cut tube (artery) and come
opposite the openings of the bulbs, but the latter, along with the
upper half of the base plate, can be rotated so that one or other
of the bulbs comes into communication with the inlet tube. In
actual practice one bulb is filled with olive oil, but in the arti-
ficial scheme air is used. When the bulbs are only half rotated
the openings are not in apposition and the fluid goes by a smg,ll
by-pass tube from the inlet to the outlet tube. Bring the empty
bulb into communication with the inlet tube at an observed
instant of time ; when that bulb has filled and the air has been
sent over into the other bulb rotate the bulbs rapidly so as to
bring the air- containing one into communication with the inlet,
and so on for a definite interval of time, say two minutes. Then
calculate the velocity as follows : — Suppose the capacity of the
bulb=5c.c., and the average time for one filling is 5 seconds,=
Ic.c. per second (=1000 c.m.m.) ; measure the diameter of the
artery (tubing), let it be say 3 m.m., then the sectional area of
the tube is (irr^) = 3.15 X (1-5)' = 7.08 sq. m.m. The total
Practical Physiology. 87
volume per second divided by this (1000 m.m. "i- by 7.08) gives
the length of the column of fluid which passes in one second=
velocity, in this case about 140 m.m., or .14 metre. The velocity
varies with each heart beat and by appropriate means a velocity
pulse can be demonstrated.
(180) Plethysmography. — With each heart beat a wave of
increased amount of blood is sent along the vessels simultaneously
with the ordinary (pressure) pulse and the velocity pulse. This
may be designated the "volume" pulse. It is demonstrated
as follows : — The organ or limb is enclosed in a roomy vessel
with rigid walls. The space between the walls and the organ
communicates by a tube with the recording apparatus (Marey's
tambour, piston recorder or reservoir with float) and air or fluid
transmission is used. Any change in volume of the enclosed
organ affects the writing lever by increasing or diminishing the
air or fluid in the recording part of the apparatus.
Application to Arm. — Enclose the arm in a Mosso's plethys-
mograph and make the junction with the skin airtight. Connect
the interior of the plethysmograph to a recording tambour, and
to a burette containing water. The whole apparatus must be
supported on a board suspended from a considerable height so that
it is impossible for the subject to move the arm out of or into the
plethysmograph. Clamp the tube leading to the burette, and
record the pulsations of the tambour lever in the usual way. Ask
the subject to take a deep breath, and note the effect on the
volume of the arm as a whole apart from the fine pulsations due
to the heart beat. For these large effects it is better to fill the
interior of the plethysmograph with tepid water, clamp off the
tambour tube, open the clamp on the burette tubing and note
the actual amount of water transferred during the following —
long deep respirations, clenching and relaxing the fist within the
plethysmograph, moving one of the lower limbs vigorously,
mental exertion, etc.
So far the circulation has been studied in its physical
aspects — the experiments which follow refer to the vital properties
of the heart and vessels.
(181) Expose the heart of a pithed frog and remove it
entirely, cutting widely so as to take away all the sinus. Place
88 Practical Physiology.
the heart in a watch glass previously moistened with normal
saline and note that it continues to beat. Its action is therefore
automatic, and rhythmical. It also possesses the power of
conducting the impulse to beat because the contraction spreads
from sinus to auricles and thence to the ventricle. Further it
can co-ordinate these movements for the two auricles contract
simultaneously. Cut through the ventricle midway between
the base and apex, the apical portion comes to rest but can be
made to contract rhythmically under the influence of a constant
stimulus such as placing on it a crystal of NaCl.
(182) In another frog expose the heart and count or record
the rate at which it is beating (172). By means of a glass tube
drawn to a fine point direct a current of warm expired air against
the ventricle and note whether the rate changes. Then direct
the hot air against the auricles and lastly against the sinus. It
will be found that the rate is increased only when the sinus is
is heated. One must be careful therefore not to allow spread
of the heat to this part when testing the ventricle and auricles.
The temperature of the breath may be sufficient in this experi-
ment, or the tube may be gently heated in the flame before
blowing through it.
These experiments show that the sinus is the most excitable
part of the heart, and it has already been seen that the beat
normally starts there.
(183) Conduction. — Gaskell's clamp (dem.). — The excised
heart is supported in a special ' clamp, the jaws of which can be
closed by means of a fine screw, and the auricles are attached to
one lever, the ventricle to another. Both levers write on a
smoked drum in the same vertical line.
While merely suported it will be seen that each auricular
beat is followed by a ventricular contraction. Now approximate
the jaws of the clamp so as to compress the tissues in the auriculo-
ventricular groove. At a certain point there will be two or more
auricular beats to each ventricular one.
(184) Chemical conditions necessary for the Heart Beat
(dem.) — An excised heart is tied on to the end of a two-wav
Practical Physiology. 89
canula, i.e., a double tube. A special normal saline (Locke's
fluid) is allowed to flow into the heart from a small reservoir
kept at a height of one to two feet, the fluid escapes during con-
traction by the other channel in the canula. The heart is then
to be placed in a Schafer's heart plethysmograph and the
contractions recorded on a horizontal drum. In this way saline
solutions of varying composition can be experimented with, and
it is found that the best results are got with a fluid which contains
besides the sodium chloride, potassium and calcium ions, and
some dextrose.
(185) Stimulation of the rhythmically beating heart. — Take
a normal cardiogram of a frog's heart (172), and while it is beating
apply a single strong break shock (101) at various phases of the
ventricular contraction. Apply the electrodes to the ventricle
by fixing them in position with a pin ; do not attempt to hold
the electrodes against the heart. It will be found that if the
■ stimulus reaches the ventricle between the beginning and the
maximum of its contraction, no result follows. If the stimulus
comes in during the relaxation an "extra systole" may result
after which the heart makes a "compensatory pause."
(186) Stannius' First Ligature. — Pith a frog — expose the
heart and arrange the apparatus as in (172) so as to obtain a
normal cardiogram. Before taking a tracing pass a ligature
under the two aortic arches and bring it round below the ventricle
so that when tied it will constrict the heart at the junction of the
sinus and auricles. Put a single knot on the thread and after
taking a short length of normal tracing tighten the ligature
in the above mentioned position. If correctly applied the auricles
and ventricles come to a standstill, the sinus continues beating ;
if unsuccessful try a second ligature. The ventricular muscle
can now be stimulated in the same way as striped muscle, as
follows : — Arrange a simple stimulating apparatus consisting
of a Daniell cell and key in the primary circuit of induction
coil ; short circuit key and pin electrodes in the secondary circuit.
Fix the pin electrodes so as to touch the ventricle, magnify the
amount of movement of the writing point by bringing the attach-
ment of the thread as near the fulcrum of the lever as the
stretching of the ventricle will permit, and use a frictionless
90 Peactical Physiology.
writing point and very lightly smoked paper. Try the effects
of stimuli of different strengths and record the height of the
contractions (102). Do not stimulate more frequently than at
ten seconds intervals. It will be found that if the ventricle
contracts at all it contracts to its maximal extent. It does not
give minimal and sub-maximal effects.
(187) On the same preparation try the effect of an eflficient
stimulus repeated at intervals of five seconds or less, moving the
drum a short distance after each stimulation. The first few
contractions are successively higher and higher — this is known
as the "staircase" effect.
(188) Put the drum in the primary circuit and take the curve
of a simple cardiac contraction as in (105). Mark the point of
stimulation and the lengths of the periods of contraction and
relaxation. Take time tracing with tuning fork.
(189) Eeturn to the arrangement used in (186), (187), but
use the Neef's Hammer, and, while the drum is revolving and
the lever is tracing an abscissa, stimulate. It will be found
impossible to obtain more than a very incomplete tetanus of
the muscle, due to its long ' 'refractory' ' period.
(190) Stannius' Second Ligature. — Tie a ligature round the
heart at the junction of the auricles and ventricle. It will be
found that the ventricle resumes its rhythmic beating but soon
comes to rest.
(191) The Cardiac Nerves. Influence of the vagus on the
heart of Mammals (dem). — Several procedures may be adopted
to show that stimulation of the vagus causes cardiac inhibition.
(a) A fine needle on end of a straw lever may be pushed into the
animal's heart through an intercostal space ; (b) the blood
pressure may be recorded ; (c) the thorax may be opened and
the heart exposed to view in an animal kept alive by artificial
respiration from an air pump.
By any of these methods it may be shown that stimulation
of the peripheral end of the cut vagus causes slowing, and then
Practical Physiology. 91
cessation of the heart beat. On removing the stimulus the
heart beats more vigorously than before.
In (c) where the thorax is opened the accelerator nerves
may be stimulated where they surround the subclavian artery.
(192) Cardio-inhibitory mechanism in the Frog. — The heart
of the frog may be inhibited by stimulation of (a) the origin of
the vagus in the medulla oblongata ; (b) the trunk of the vagus ;
(c) the sino-auricular junction (intracardiac inhibitory nerves)
Decapitate a frog by cutting transversely through the
upper jaw and skull in line with the anterior margins of the
tympanic membranes, then pith the spinal cord entering the pin
about an eight of an inch below the usual spot — junction of skull
and vertebral column. The portion of the brain left between
these two points includes the optic lobes and medulla oblongata
from which latter the vagi arise.
Lay bare the heart and prepare to record the movements
by the usual suspension method (172). Fit up a tetanising
apparatus (Daniell cell, simple key, Neef's Hammer in primary
circuit ; short circuit key and pin electrodes in the secondary).
{a) Insert and fix the pin electrodes in the exposed cranial
cavity so that one electrode lies on each side of the medulla.
Then while a normal tracing is being recorded stimulate with
the interrupted current at first with the coils at such a distance
apart that the stimulus is just perceptible when applied to the
tongue. If this fails to produce an effect, gradually increase
the strength of the stimulation. Mark on the tracing the points
where the stimulation was applied, and where it ceased.
(6) Isolate the vagus nerve. Three nerves emerge from
the skull near the angle of the jaw in the frog — the glosso-
pharyngeal, the vagus, and the hypoglossal. The glosso-
pharyngeal and the hypoglossal are easily distinguished, both
being comparatively superficial and both turn upwards along
the floor of the mouth. It is advisable to dissect both these as
far back as possible and then cut them away. The vagus will
be found close to the carotid artery, crossing the base of the
petrohyoid muscle. Its dissection is facilitated by placing a
thick glass rod in the oesophagus. The nerve is accompanied
92 Practical Physiology.
by its laryngeal branch wMch is relatively large and apt to be
mistaken for the main trunk ; but the branch Ues on the upper
side of the carotid artery and can be traced towards the larynx,
while the vagus itself is on the lower side of the artery. Pass
a thread under the nerve, raise it gently, apply the electrodes
and stimulate with the interrupted current (Neefs Hammer).
There may result either acceleration or inhibition of the heart's
action due to the fact that the vagus contains both cardio-
accelerator (sympathetic) fibres, and cardio-inhibitory fibres.
Weak stimulation has more effect on the inhibitory fibres,
while strong stimulation effects the accelerators more.
(c) Fix the pin electrodes against the heart in such a positioi;i
that the pin points touch the white crescentic line which marks
the sino-auricular junction, and again record the effect of stimu-
lation. The pin-points should be close together.
(193) Effect of some Drugs. — Paint the sinus with a dilute
solution of Pilocarpine, or of muscarine using a camel hair
brush. The heart stops beating in diastole. Now apply a solution
of atropine in similar fashion, and the heart wUl resume beating
While the heart is under the iirfluence of the atropine repeat (a)
(6) or (c) of preceding experiment. No inhibition results because
atropine paralyses the terminations of the vagus in the heart.
Acceleration may be obtained if the trunk of the vagus is stimu-
lated due to the uncomplicated effect of stimulation of the
sympathetic fibres contained therein. On the same frog after
thoroughly washing away the atropine, or on a fresh frog, the
effect of nicotine may be tried. This drug paralyses nerve
fibres where they end in connection with other nerve cells in
ganglia. Suppose inhibition had been obtained by methods
(ffl), (6), and (c), above before the application of nicotine, it will
be obtained only by method (c) after painting nicotine on the
heart, because in (c) the cardio-inhibitory fibres are stimulated
after they have passed their cell stations.
(194) Effect of Bile on the beating Heart.— While a normal
cardiogram is being taken in the usual way, dissect out the frog's
own gall bladder without allowing the contents to escape. Hold
it over the heart and cut it open so that the ventricle is anointed
with bile. Note the slow weak beats which follow.
Practical Physiology. 93
(195) Innervation of the Vessels. — Examine the vessels in
the ears of a rabbit which has had its cervical sympathetic nerve
divided on one side. Note the dilated condition of the vessels
on that side and the absence of the slow rhythmical changes
which occur normally. If the animal becomes angry, the vessels
of the ears dilate, but those of the sound side become more dilated
than those of the side operated on, due to the intact vaso-dilator
fibres on that side. (Note also in this animal that the pupil on
affected side is contracted, and the eye as a whole retracted).
(196) Perfusion of Blood Vessels.— Pith a frog ; expose the
heart and aorta ; make a V-shaped incision in one of the aortic
arches ; insert and tie in a canula connected to a small reservoir
of Ringer's saline solution ; see that the canula is full to the point
before inserting it. Tie the other aortic arch ; suspend the frog
vertically ; cut the sinus open and bring the toes together so
that the fluid which perfuses the whole circulatory system and
which escapes at the sinus may drip from the toes. Count the
number of drops per minute ; add to the perfusion fluid some
drug capable of affecting the calibre of the arteries, and again
count the rate. Adrenalin is a good substance to use here.
CHAPTER VII.
RESPIRATION.
Examine microscopical preparations of trachea, bronchi,
lungs, pleura, etc.
(197) Graphic record of respiratory movements. — The move-
ments of the thoracic walls during inspiration and expiration are
recorded by means of a stethografh consisting of a tambour and
writing lever connected to a flexible indiarubber tube fitted
to the inside of a leather belt. The belt is secured'' round the
chest a little below the level of the nipple, and the movements
of the lever are recorded on a slowly moving blackened drum.
Record also the efiects of yawning, coughing, sneezing, and,
if it can be obtained, hiccup.
(198) Ratio of frequency of respiration to pulse rate. — Deter-
mine this by counting each in the sitting and standing posture, and
after running once or twice up and down stairs. The usual ratio
is 1 to 4 or 5.
(199) Respiratory Sounds. — Listen with the stethoscope to
the sounds heard during respiration when the stethoscope is
placed over the trachea or large bronchi (between the soapulte
at the level of the fourth dorsal vertebra) — bronchial or tubular
breathing, and in the axillary region, angle of scapula, or apex of
lung — vesicular breathing. In bronchial breathing a sound is
heard during both inspiration and expiration ; the sounds are
the same in quality of tone, are separated by a distinct silent period
at the end of inspiration and beginning of expiration, and they
Practical Physiology. 95
occupy practically the whole duration of each movement. Vesi-
cular breathing, on the other hand, consists of two sounds which
differ in character, the expiratory part being low in pitch while
the inspiratory sound has a characteristic rustling sound ; further,
there is no distinct interval between the sounds, the inspiratory
sound occupies practically the whole period of inspiration and is
followed immediately by the expiratory unless the subject voluntar-
ily holds his breath at the end of inspiration. Of great practical
importance is the fact that in healthy vesicular breathing the ex-
piratory sound is only about half as long as the inspiratory. In
children, however, the sounds are equal in duration and have a
harsher tone than in the adult. Note also the ' 'vocal resonance."
Ask the subject to repeat " one, one, one," or " ninety-nine,"
and note the intensity of the buzzing sound usually heard.
(200) Spirometry. — The spirometer is used to determine the
volume of air that can be expired after the fullest possible in-
piration = vital capacity. Two forms are in common use :
Hutchinson's on the principle of a balanced gasometer, and various
forms in which the current of expired air causes revolutions of
vanes with which a series of indicators are connected as in the
common household gas meter. Determine your vital capacity
with each form of instrument.
(201) Intrapulmonary Pressure. — Connect a mercurial man-
ometer to a mouthpiece by indiarubber tubing and determine the
maximum expiratory pressure after a full inspiration has been
taken. Estimate the inspiratory pressure in the same way, but
be careful not to use suction.
(202) (Dem.) Effect of division of the vagi on the respira-
tory movements.
(20-3) (Dem.) Observe the symptoms produced in a small
animal placed in an air tight chamber.
(204) Chemical changes in respired air. — Determine the
amounts of CO 2 and of Oj in expired air collected in a Hutchin-
son's spirometer. The COg is absorbed by means of KOH, the
O2 by pyrogallate of potash in some form of gas analysis apparatus
96 Practical Physiology.
such as that devised by Haldane. The amount of COg in expired
tiir may also be determined approximately by the following
method ■ — First find the capacity of the ungraduated lower end
of a burette between the lowest mark (50) and the clip. This may
be done by running in water from another burette. Now fill the
burette with water, invert it and place it in the tall jar of water
without allowing air to enter. Fill it about three-quarters full
of expired air by means of the bent glass tube the lower end of
which is to be inserted into the open end of the burette.
Read the volume of expired air which has been obtained
being careful not to hold the burette in the hand ; hold it by the
indiarubber tubing or use a cloth between your hand and the glass.
The water level inside the tube must be the same as that outside
it when the reading is made. Now bring the open end of the
burette near to the surface of the water and insert a small piece
(say half to one inch long) of stick NaOH. Remove the burette
from the jar after closing the lower end with a small cork. Shake
to and fro so that the CO 2 may be absorbed by the solution of
caustic soda. After about five to ten minutes replace the burette
in the jar, uncork it, and again read the volume of the air with the
water inside and outside the burette at the same level and cal-
culate the percentage diminution of volume due to absorption
of the CO 2.
For a method (Haldane-Pembrey's) of estimating the
respiratory interchange in small animals see exp. 305, H.
(.205) The gases of blood. (Dem.) By means of a special
form of mercurial pump (Leonard Hill's) the gases -of a sample of
venous or arterial blood are collected and analysed in a. similar
way.
Haldane and Barcroft's apparatus for estimating the O2
and CO2 in a small sample of blood by the ferricyanide method
for O2 and tartaric acid for CO 2 may also be demonstrated.
(206) The oxydases or oxidizing ferments in the tissues. —
Add a drop of fresh blood from the finger to a few c.c.'s of ozonic
ether or watery hydrogen peroxide in a test tube. An evolution
of gas will be seen, due to the liberation of oxygen from the
H2O0 by the oxydases present. The same ferments are said
to be the cause of the Guaiac Reaction (159) of blood and extracts
Practical Physiology. 97
of tissue, but the question is complicated by the presence of sub-
stances which act as catalytic agents and which withstand boiling,
thus blood gives the guaiac reaction after boiling due it is believed
to the stroma of the red corpuscles acting in this way.
(207) Artificial respiration. — Demonstration of Schafer's
Prone-pressure method, which consists in laying the subject in
the prone position, preferably on the ground, with a thick folded
garment under the chest and epigastrium. The operator puta
himself athwart or at the side of the subject facing his head,
and places his outspread hands on each side over the lower part
of the chest below the scapulae. He then slowly throws the
weight of his body forward, so as to bear upon his own arms, and
thus press upon the thorax of the subject and force air out of the
lungs. This being affected, he gradually relaxes the pressure
by bringing his own body up again to a more erect position with-
out moving his hands. Repeat the movements twelve to fifteen
times a minute
CHAPTER VIII.
ALIMENTARY SYSTEM.
Examine microscopical preparations of parts of the alimen-
tary tract — palate, tongue, tooth, tgnsil, gullet, stomach, intest-
ines, and of the related glandular organs — salivary glands,
pancreas and liver.
(208) (Dem.) Observe the intestinal movements in a rabbit,
the abdomen of which has been opened in a bath of warm normal
saline, also the means of recording the movements and the influence
of section and stimulation of nerves.
(209) The contraction of non-striped muscle. — Cut a trans-
verse or oblique ring of stomach wall of frog. Arrange it on the
myograph stand using a straight lever with a frictionless point
as in heart work (Fig. 14). Fix a pair of pin electrodes in position
so as to touch the strip of tissue, connect these through a simple
key to one or more Daniell cells, and take a tracing on the diura
of the contraction wave of the non-striped muscle. The length
of the latent period is appreciable, and can be measured by putting
the drum in the circuit and so obtaining the point of stimulation.
Galvanic electricity gives better results than faradic in this kind
of tissue and the pins of the electrodes should be as widely separ-
ated as possible, or the current should be arranged to pass through
the whole length of the strip. Spontaneous contractions may
also occur in the strip.
Practical Physiology. 99
CHEMISTRY OF THE DIGESTIVE SECRETIONS.
(A) SALIVA. — Collect some saliva and filter it. Note the
reaetioii to litmus paper — alkaline.
(210) Add a few drops of acetic acid — precipitate of mucin
in the form of a stringy coagulum results. The precipitate is
insoluble in excess of acetic acid.
(211) Filter off the mucin and test the filtrate for protein;
result — negative.
(212) Add some ammonium oxalate to some of the same
filtrate — clouding, due to the presence of calcium appears.
(213) The saliva of many persons contains alkaline sulpho-
cyanides. Test for this by adding a drop of HCl to some saliva,
filter off the mucin, and to the filtrate add a small amount of
dilute ferric chloride — a red colour appears if KCNS is present.
(214) Ptyalin — the digestive ferment of saliva is tested for
by its action on boiled starch. Have ready some saliva, boiled
starch mucilage, a water bath heated to 40°C., a series of small
drops of iodine solution arranged regularly on a glazed white
porcelain slab. Add to some starch mucilage in a test tube a
small quantity of saliva, mix, and place the tube in the warm bath.
As digestion proceeds test the contents of the tube from time to
time by taking out a drop of the mixture on a glass rod, placing
it alongside one of the iodine drops and allowing the two to mingle.
The first result of ptyalin action is to change the starch mucilage
into soluble starch or amidulin ; this is indicated by a diminution
in the opacity of the mucilage, the reaction with iodine being still
a blue. Then later there appears erythrodextrin which gives a
red colour with iodine ; later still the mixture gives no colour with
iodine but contains a dextrin which can be precipitated by alcohol
or other means — achio5-dextrin. The final result is the reduc-
ing sugar, maltose ; but some maltose is formed early in the diges-
tion, and under artificial conditions where the products (dextrins
and maltose) are allowed to accumulate there always remains
some unchanged dextrin (achroo-dextrin). Test the digested
100 Practical Physiology.
mixture for a reducing sugar by Trommer's and Barfoed's tests.
To another portion add some alcohol and note whether a precip-
itate appears. {Vide ante — Polysaccharides). It will be noticed
that the action of ptyalin is similar to the action of dilute HCl
at boiling temperature, but the end product in the latter case is
gluccse due to the maltose being further hydrolysed.
(215) To some starch mucilage add some saliva which has
been boiled and cooled. Keep at 40° C. No digestion occurs
because all ferments are rendered inactive by boiling.
(B) GASTRIC JUICE.— This secretion contains free mineral
acid (HCl) in a fairly definite proportion. A solution of HCl is
put out similar in strength to that usually secreted. Test it
as follows : —
(216) To ordinary blue litmus paper it reacts strongly acid.
Congo red paper is turned blue, this indicates that the acidity is
due to free acid, not to an acid salt such as sodium dihydiogen
phosphate. Dry the piece of blued Congo paper, extract it in
a dry test tube with ether — the blue colour remains (cf. 221).
(217) Gunzburg's Test. — (Reagent =^ 2 parts phloroglucinol,
1 part vanillin in 30 parts absolute alcohol). Add a few drops
of this reagent to a similar amount of the acid solution in a
porcelain basin and evaporate gently to dryness over a small
flame. A carmine rod colour indicates the presence of free
mineral acid.
(218) Boas' resorcin test. — (Reagent = Resorcin 75 grains,
white sugar 45 grains, dilute spirit 3J ounces). Use in same way
as for Gunzburg's teat. Result — a purple colour if free mineral
acid is present.
(219) The Trofoeolin Test (also known as Boas' test).
Evaporate to dryness a mixture of equal parts of a saturated
alcoholic solution of Tropaeolin 00 and the suspected fluid —
lilac to violet glancing colour appears if free HCl present.
Practical Physiology. 101
(220) Estimation of the strength of acid present. — Measure out
exactly, in a graduated pipette or burette, ten or twenty c.c. of
the acid fluid into a small flask or beaker, dilute with some water,
add a couple of drops of phenol-phthalein solution, run in some
tenth-normal alkali from a burette noting the starting point and
the end point ; the latter is taken when the whole fluid assumes
a very faint pink colour, remaining permanent for one minute or
so ; and then calculate the amount of acid present in 100 c.c.
Each c.c. of tenth-normal alkali exactly neutralises one c.c.
of tenth-normal acid., so that each c.c. of y^ soda used = 3.65
mlgm. HCl. (Normal HCl ^ 36.5 grm. per litre, tenth-normal
= 3.65 grm. per litre == 3.65 mlgrm. per c.c.) Suppose 8 c.c. of
■/„ neutralises 10 c.c. of the acid fluid then the amount of HCl in
the ten c.c. is 8 X 3.65 mlgrm. = 29.2 mlgrm. = .0292 grm. = 0.292
per cent.
The normal percentage of HCl present is 0.2%.
Abnormal acidity. — In some pathological states the stomach
contains other acids than HCl, and it is of great practical
importance to distinguish between these organic acids and the
mineral acid (HCl). Lactic Acid is one of the commonest of
these abnormal acids, but acetic and butyric acids also occur.
(221) Apply the following tests to a weak lactic acid solu-
tion : —
Litmus is affected in the usual way.
Congo-red Paper turns a bluish violet, and after drying, the
colour can be extracted with ether, leaving the paper
red.
Boasts Test (218) does not give a purple, neither does
Gunzburg's give red.
Uffelmann's Reagent (120) gives a yellow colour, as with
sarco-lactic acid. HCl completely discharges the
colour of the reagent.
In many cases, however, both HCl and organic acids are
present, and their separation must be affected. There are various
good laboratory methods for estimating the relative amounts of
102 Practical Physiology.
these, but the following simple procedures will be sufficient in
most cases. Do these on a mixture of equal parts of the dilute
HCl and lactic acid given you or on gastric contents from a
suitable case : —
(222) The colour of Uffelmann's reagent will probably be
completely discharged because of the action of the stronger
inorganic acid. To about one-third full of a large test tube of
the mixture add an equal amount of ether, cork and shake,
holding the tube in a cloth while doing so. The ether extracts
the lactic acid, and if the ethereal layer is carefully poured ofi
into a porcelain basin containing a few c.c.'s of hot water the
ether evaporates leaving the lactic acid. Evaporate off all the
ether on a water bath and test the watery residue for lactic
acid as in (221).
(223) Pepsin. — Prepare an artificial gastric juice from the
mucous membrane of the pig's stomach by dissecting off the
mucous membrane, mincing it, adding some 0.2% HCl and allow-
ing it to digest for some hours. The fluid is then allowed to
cool, and is filtered after standing for one to two days.
(224) To some of this artificial gastric juice add some raw
meat fibre or washed fibrin, or a fine emulsion of partially coagul-
ated egg white and note the changes which occur when the mixture
is incubated at 40° C. The insoluble protein becomes swollen
and gradually dissolves.
(225) Proceed to examine a gastnc digest, either the one
above mentioned or one similarly prepared and allowed to digest
for several hours. First take the reaction to litmus, it is acid ;
add diluted KOH till the solution is almost neutral, acid albumin
will be precipitated, if the KOH has been added in too great amount
the acid albumin will be redissolved (41), but can be again pre-
cipitated by adding acetic acid in slight excess ; filter off the
precipitate of acid albumin if any is present and boil the filtrate
to remove unaffected ooagulable proteins ; filter again if necessary
and cool the filtrate. (The bodies so far obtained, coagulable
protein and acid albumin, might result from the action of the
weak acid at 40° C. and do not necessarily indicate the presence
of active pepsin). Half saturate one half of the last filtrate
Practtcal Physiology. 103
with ammonium sulphate. A precipitate of primary albumoses
may result ; filter again, using a dry filter paper and funnel (keep
the filter paper with this precipitate of primary albumoses) ; if
primary albumose has been found, test the other half of the fil-
trate with nitric acid, a precipitate will appear which clears up on
heating and reappears on cooling. The filtrate from the primary
albumose precipitate is now to be tested for secondary albumose
and peptone as follows : — Fully saturate the fluid with crystals
of ammonium sulphate, shaking thoroughly and warming gently
to secure complete saturation — a precipitate will indicate the
presence of secondary albumose, filter and test the filtrate for
the presence of protein by the biuret test (17) ; since peptone is
the only protein which is not precipitated by full saturation with
AmS04 a positive result with the biuret test indicates its presence
in the filtrate, and the pink colour of the reaction is also character-
istic ; in doing the biuret test with a fluid containing AmS04
use very concentrated KOH, see (25). The precipitate of pri-
mary albumoses mentioned above is to be washed with half-
saturated ammonium sulphate, then dissolved in water, and
alcohol is to be added till the total volume is three times that of
the watery solution ; the concentration of alcohol will then be
about 6fi%, and in this strength one of the varieties of primary
albumose, viz., the heteroalhumose, is precipitated while the other
frotoalhumose remains in solution. These varieties of albumose
can also be separated by dialysis, which causes precipitation of
the hetero variety only.
The following scheme summarises the method of examining
a digest (peptic or tryptic)
Neutralise precipitate of albuminate (acid or alkali
albumin.)
Filter if necessary and boil coagulation of unaltered
protein.
Again filter if necessary and half -saturate with AmSO^
precipitate of primary albumoses.
Again filter if necessary and complete the saturation with
AmSO^ precipitate of secondary albumose.
Again filter and test with Biuret pink colour in-
dicates peptone.
104 Practical Physiology.
(225a) In examining the contents of a stomach after a test meal
both pepsin and rennin should be examined for. This is
most readily done by neutralising the fluid with CaCOa ;
filter ; add some of the filtrate to fresh milk and keep at
40° C. — coagulation will result if rennin is present ; the
remainder of filtrate is to be mixed with finely emulsified
egg white and HCl so that the percentage of the latter may
be about 0.2 (7 c.c. of concent. HCl to the litre), the mixture
is then incubated at 40" C. for some hours, neutralised with
CaCOa filtered, and the filtrate may be completely saturated
with AmSOi, filtered, and the new filtrate tested for
peptone, or the neutralised filtrate may be boiled, filtered,
and this filtrate tested for albumose by the nitric acid test.
This test for albumoses is a most useful one, and is chietij'
due to hetero albumose.
In examining vomited matter it is frequently important to
ascertain if the fluid contains blood. If so, the haemoglobin
has usually been converted into haematin, giving rise to the
appearance of "coffee grounds" ; obtain some of these by
filtering or centrifuging, add KOH, heat gently, cool, add
ammon. sulphide and examine spectroscopically for haemo-
chromogen( 166), or the test for haemin (168) may be applied
to the brown substance.
(226) Coagulation of milk by rennin is another fermenta-
tive action produced by gastric juice. Add to a test-tubeful
of milk a few drops of commercial rennet (which is usually an
extract of the stomach of a calf), or the artificial gastric juice
made from the stomach of the pig (223) may be used if neutralised,
otherwise the HCl present will precipitate the caseinogen of
the milk. Keep the test tube at body temperature, a coagulum
forms which contracts and squeezes out whey. Test boiled rennet
in the same way. Calcium Salts are necessary : — to another
test tube of milk add a few drops of a soluble oxalate, mix, add
rennet, incubate — no coagulation results ; now add CaCU in
amount sufficient to combine with the oxalate and leave some
calcium ions free, re-incubate — coagulation occurs.
(C) PANCREATIC JUICE.— As in the case of the gastric
juice, a watery or glycerine extract of pancreas contains the chief
ferments. They are four in number : trypsin, amylopsin,
steapsin, and a milk-coagulating ferment.
(227) Trypsin is not present as such in the pancreatic juice
but as the zymogen — trypsinogen — which is activated to trypsin
by the enterokinase of the intestinal juice ; a similar change
Practical Physiology. 105
occurs when a pancreas is allowed to lie exposed to the air, so
that extracts of the gland about a day after death contain active
trjrpsin. If the gland is treated as described in (74) for pre-
paration of its nucleo-protein, i.e., extract for twelve hours with
ammoniacal water, strain, precipitate the nucleo-protein by adding
acetic acid cautiously, filter and dissolve the precipitate of nucleo-
protein in half per cent, sodium carbonate — it will be found that
this solution contains tr)rpsin, possibly because it has adhered
to the precipitate, for this is a characteristic of ferments that
they are apt to cling to precipitates formed in their solutions.
Test the activity of a trypsin solution prepared in this way,
or of a commercial extract of pancreas, on native protein by
allowing it to digest for several hours at 40°. The action pro-
ceeds best in the presence of alkali up to the strength of 1%
sodium carbonate ; it ceases if free mineral acid is present.
Examine the digest in the way described in (225) but for ' ' acid ' '
read ' ' alkali ' ' in relation to reaction and the form of meta-
protein which results.
(228) Add some thymol to the remainder of the digest and
allow it to incubate for several weeks to obtain the ultimate
products (amido acids, etc.) which result from the tryptic digest-
tion flus the action of erepsin derived from the tissue of the
prancreas. The ultimate products most easily recognised are
leucin and tyrosin which form characteristic crystals and trypto-
phane, which gives a violet colour on addition of water saturated
with bromine, after acidifpng.
(229) Amylopsin. — Test the action of a watery extract of
fresh or of dried pancreas on starch mucilage in the same way as
for Ptyalin (214) ; the products of digestion are the same.
(230) Steapsin — Make a concentrated watery extract of
fresh pancreas ; mix some of it with five to ten times its bulk of
cream or milk ; add a few drops of chloroform, a few drops of
litmus solution, mix and if the reaction is acid (red colour) add
a trace of sodium carbonate so as to have the mixture just alkaline
and not excessively so. Divide this mixture into two ; boil one
portion ; keep both at 40° C. till next day, and if the steapsin
has acted, the unboUed fluid will show a red colour, or more red
106 Practical Physiology.
as compared with the boiled portion, due to the formation of
free fatty acid from splitting of the fat. It should be unnecessary
to explain the reasons for the above procedure ; the student
ought to work out for himself the reasons for boiling one of the
tubes, for adding chloroform, for using cream preferably to milk,
for making the mixture before dividing into two, etc.
(231) Pancreatic juice and extracts cause coagulation of
milk. Test this on fresh milk as in (226).
(D) BILE. — Bile may be included here among the digestive
juices although it of itself has no marked digestive action on the
food stuffs ; it aids the action of pancreatic juice however.
(232) Test the reaction of ox bile to litmus paper ; it is
alkaline.
(233) Mucin. — Add a few drops of acetic acid — a precipitate
of mucin is thrown down.
(234) Bile Salts— Peiiemfco/er's Test.—'Yo some diluted bile
or to the filtrate from the mucin precipitate (233) add a few
crystals of cane sugar, and shake to accelerate solution and to
cause a froth to appear ; a.llow some concentrated sulphuric acid
in excess to flow down the side of the tube — a purple colour
develops where the acid comes in contact with the bile-and-sugar
mixture ; this colour is also well seen in the froth where it has
been touched with the acid ; now mix the contents of the tube
and regulate the temperature of the mixture to about 70° C
by cooling under the tap or heating over the bunsen — the purple
colour ought now to become evident throughout the mixture and
if cooled and diluted with alcohol a characteristic spectrum will
be seen when examined with the spectroscope ; it consists of one
band between D and E, near E, and one near F. The rationale
of the test is that the strong HgSO^ produces furfurol from the
sugar, and at the same time, cholalic acid from the bile salts, and
the interaction of these two produces the purple colour. If too
much sugar is added, the charring which results (54.) obscures the
result. Furfurol solution (0.1%) may be used instead of sugar.
This colour reaction is analogous to some protein colour reactions
Practical Physiology. 107
{e.g., glyoxylic (18) and Molisch's (20) where a colour results
from the interaction of an aldehyde and a substance with
an aromatic nucleus, in this case furfurol (furfuraldehyde) and
cholalio acid. The test is therefore unreliable in the presence of
ordinary proteins.
(235) Bile Pigments.— 6remZm's Test. — To some diluted bile
or bihous urine add some impure nitric acid so as to form a deep
layer at the bottom of the test tube. At the plane of contact a
series of colours appears, spreading up into the bile ; (at the same
time the mucin is precipitated if bile itself is used). The colours
result from the oxidation of the bilirubin and biliverdin of the bile,
and occur in the following order from above downwards, bili-
rubin (red), biliverdin (green), bilicyanin (blue), choletelin (yellow)
or if concentrated (reddish). The reaction may also be done on
a white porcelain plate by allowing a little bile to spread out on the
plate in a thin layer and placing a drop of impure nitric acid in
contact with it.
(236) Sulphur Test for Bile. — Bile salts can reduce the sur-
face tension of a watery fluid so that fine grains of sulphur will
sink through the fluid. Take two test tubes, one of water and
one of diluted bile ; add to each a few fine particles of powdered
sulphur. The sulphur sinks readily through the bile but remains
floating on the water.
(237) Cholesterin — previously considered as a constituent
of protoplasin in general, occurs in relatively large amount in
bile, and in some diseased conditions forms a gall-stone. Other
forms of gall-stone consist chiefly of lime and pigments. Examine
the appearances of some common gall-stones — note that the
cholesterin ones are light and float in water. Extract some pow-
dered gall-stone with alcohol on a water bath ; allow a few drops
of the extract to evaporate on a slide, and examine under the
microscope for crystals of cholesterin. Repeat the miorochem-
ical test (75) with H2SO4 and iodine. If much cholesterin is
present make a chloroform extract and repeat Salkowski's
reaction (76) and (77).
(238) Bile has a slight power of reducing cupric hydrate
solutions.
108 Practical Physiology.
(239) One or other method of isolating the bile salts may be
demonstrated while bile is being studied in the class.
(240) The inorganic constituents of bile consist of the usual
chlorides, phosphates, sodium, potassium, calcium, etc. Iron
occurs only in mere traces.
Action of bile in digestion.
(241) On Proteins. — Bile precipitates and is partially pre-
cipitated by the constituents of a gastric digest (chyme). Test
this statement ; the mixed precipitates consist chiefly of acid
albumin, mucin and bile salts. , Peptone is used clinically by some
observers as a test for bile salts in urine.
(242) On Carbohydrates. — Bile aids amylopsin in the diges-
tion of starch. Take some very thin starch mucilage, add some
amylopsin, divide into two portions, to one add some bile, and
place both on the water bath at 40° C. Test at intervals a drop
from each tube against a drop of iodine on a porcelain slab, as in
(214) and note the relative time taken by each to show the changes
in the starch.
(243) On Fats. — Bile aids the emulsification and splitting of
fat. Compare the permanency of the emulsion formed by shaking
the following mixtures : —
(a) Water and oil {rancid olive oil), (fe) 1% sodium car-
bonate and oil, (c) 1% sodium carbonate and oil flus
some bile. In (a) no real emulsion forms, in (&) a
slight emulsion depending on the amount of soap
formed if the oil is rancid, in (c) a good emulsion
forms.
(E) SUCCUS ENTERICUS contains the ferments, Invertin,
Maltase, Lactase, Erepsin, and Enterokinase.
(244) Invertin. — Test the efiect of a watery extract of intes-
tinal mucosa on the inversion of cane sugar, at 40°C. If
inverted, the cane sugar yields glucose which will reduce
Fehling.
Practical Physiology. 109
(245) Test the effect of the same extract on a solution of
maltose. If Maltase, the maltose-inverting ferment is present,
Barfoed's reagent will be reduced by the products (44).
(246) Test the effect of the same extract on lactose. If
Lactase is present the lactose is inverted, yields glucose which
will give the fermentation test (50). Do control experiments
with boiled extract of the mucosa in each case.
(247) Erepsin. — Add some of the watery extract of intes-
tinal mucosa to a solution of albumoses and peptones so diluted
that it gives a faint but distinct biuret reaction. Keep the mix-
ture at 40°C and test from time to time. If the erepsin is present
in small amount, the result (disappearance of the biuret reaction)
may take some days to appear cf. (228) ; control as above.
CHAPTER IX.
THE URINE.
The amount of urine passed daily varies considerably, but
is usually 40 to 50 fluid ounces (or about 1500 c.c).
The colour varies from light straw to amber, according to
the concentration. It is due to certain definite pigments —
urochrome, urobilin, hsematoporphyrin, etc.
(248) The reaction is generally acid to litmus paper due to
acid salts (NaH2P04.) as shown by th.e fact that it does not afiect
Congo red paper (c.f. (216) gastric juice). It may be amphoteric,
neutral, or even alkaline during active digestion. Decomposing
urine and normal herbivorous urine is alkaline.
(249) The specific gravity varies from 1015 to 1025, or less,
or more according to the amount of water present. It is usually
taken with a urinomeler — a hydrometer specially graduated
from 1000 to lOflO. Eemove any froth, that may be present
and allow the urinometer to float freely in the urine without
touching the sides of the jar. Read the level at which the
instrument floats. In light coloured urines this may be done
conveniently by looking through the fluid, with the eye almost
on a level with the surface ; in this way the error arising from the
meniscus which forms where the urine is in contact with the stem
of the hydrometer may be avoided. The urinometer readings
vary with temperature and are correct only at the usual room
temperature (15° C). For each 3° above or below that tem-
perature add or subtract a unit to the last figure of the reading,
e.g., if the temperature of the urine is 18°C. and the apparent
specific gravity by the urinometer 1016, the correct specific
gravity is 1017.
Practical Physiology. Ill
CHEMICAL COMPOSITION.— The chief organic constituents
of urine are — urea, uric acid and xanthin bases (alloxur bodies),
creatinine, hippuric acid, ethereal sulphates, and pigments, with
variable traces of other substances.
The inorganic constituents are sodium, potassium,
ammonium, calcium, magnesiuiu, partly free as ions and partly
united to the acid radicles as chlorides, phosphates, sulphates,
carbonates, etc.
The chief substances which appear fathologicalhj in urine
are albumin (and globulin), blood (corpuscles and pigments),
bile (salts and pigments), sugar (glucose chiefly and the acetone
bodies) ; and there may he, an abnormal increase or diminution
of the amount of water passed, or of a normal constituent, e.g.,
increase of uric acid and urates, of ethereal sulphates, or of phos-
phates, etc. ; or a diminution of urea, or of chlorides, etc.
(A) ORGANIC CONSTITUENTS.
Urea C0(NH,,)2. — Urea is the chief solid found in urine.
(250) Examine the crystals of pure urea. Note the cooling
sensa.tion when they are applied to the tonaue duo to their rapid
solution ia water. Note the great solvhilily of urea in water,
and in alcohol, and its insolubility in anhydrous ether and
chloroform.
(251) In concentrated solution it forms additive com-
fovnis with acids. Test this by adding a drop of concentrated
watery solution of urea to a drop of pure HNO3 on a slide ;
examine the crystals of nitrate of urea which form. Proceed
similarly with a concentrated solution of oxalic acid (oxalate of
urea). The crystals take the form of rhombic plates in each
(252) Heat some urea crystals in a dry test tube. They
melt and ammonia is evolved (test with moist red litmus paper).
A sublimate forms on the colder upper part of the tube, consisting
chiefly of ammonium carbonate. Heat till the contents of the tube
become solid, then cool, add a few drops of water, a few drops of
112 Peactical Physiology.
KOH solution and a drop of watery CUSO4. solution. A fine pink
colour appears, due to the presence of biuret. (Proteins give
a similar reaction on adding KOH and CuSO^, and the same
name is generally applied to the reaction, but the atomic grouping
which gives the colour is not quite the same in the two cases).
(253) Add some KOH to urea crystals and heat — ammonia
is evolved (test with moist litmus) cf. action of alkali and heat
on protein (34).
(254) Some oridising agents such as sodium hirpo-bromite
(NaBrO), and nitrous acid decompose urea, liberating nitrogen
and COo. To some urea solution in a test tube add a few drops
of sod. hypobrom. — A copious evolution of gas results. The
reaction is expressed by the formula : —
3 NaBrO -f CO (NHa)^ = 3 NaBr -f 2H2O -f 00^ -f N^
since the solution of hypobromite is strongly alkaline, the CO 2
is absorbed, and only the Ng is liberated. This forms the basis
of the next exercise.
(255) Quantitative estimation of Urea in Urine. — The
apparatus for this consists of a burette inverted in a tall jar
filled with water and connected by tubing to a bottle containing
a small glass tube.
Test whether the apparatus is airtight by inserting the
stopper in the bottle and raising the burette ; if there is no leakage
the column of water in the burette remains standing at a higher
level than the water in the jar.
Place 20 to 25 c.c. hypobromite solution in the bottle, and
exactly 5 c.c. of urine in the small tube ; place the tube inside
the bottle without allowing the fluids to mix ; insert the
stopper securely ; place the bottle in a jar or dish of water
to cool ; then read the level of the water in the burette with the
water inside it at the level of the surface of the water in the jar.
Now tilt the bottle so as to mix the urine and hypobromite solu-
tion and shake very gently ; try to let some of the hypobromite
get into the small tube to act on the urine that wets its inside ;
replace the bottle in the cold water ; allow it to cool ; then read
the burette in the same way as before, i.e., with the water inside
Practical Physiology. 113
and outside the burette at the same level. Since the CO 2 is
retained in the alkaline hypobromite, the increase in the volume
of the gas contained in the apparatus (the difference between
the readings of the burette) is due to the nitrogen evolved from the
urea in 5 c.c. of urine. The calculation of the amount of urea
present is made as follows : — From the formula given above
(254)1 gramme of pure urea should yield 372 c.c. of nitrogen
gas at 0°C and 760 m.m. barometric pressure ; but when the
decomposition is done under the usual laboratory conditions,
and with urine, which always contains other substances capable
of yielding nitrogen with hypobromite, the amount of gas
corresponding to 1 grm. urea is only 354.3 c.c, i.e., 0.1 grm.
would give 35.43 c.c; suppose the burette readings before and
after decomposition of the urea were respectively 48.7 and 21.3
= an increase of volume due to Nj of 27.4 c.c, this would equal
(11*7 X 0.1) gram, urea in 5 c.c. = 0.077 grm., the per-
centage is therefore 1.54, and the amount in a total day's urine
of 1500 c.c. = 23.1 grm., a somewhat low figure. Southall's
and various other ureometers used clinically will be demonstrated.
Uric Acid. — Note the shape and colour of uric acid crystals
under the microscope. The chief form is that of pointed oval
plates arranged in stars, rosettes, and barrel shapes. They are
almost always coloured with the urinary pigments. Uric acid
occurs in urine united to bases and the compound frequently
settles out on cooling.
(256) Examine such a deposit of urates and note that they
dissolve on moderate heating. Crystals of uric acid may be
obtained by strongly acidifying with HCl, such urine or even
normal urine and allowing it to stand for a day or less.
(257) Note the solubilities of pure uric acid. It is almost
insoluble in cold water (1 in 16,000), more soluble in hot (1 in 2000).
It is insoluble in alcohol and ether. It dissolves in concentrated
HoSOi,. without decomposition, and is again precipitated on adding
a drop of the solution to a test-tubeful of water. It dissolves
in alkalis and alkaline carbonates forming urates.
(258) Make a small amount of a concentrated solution of
urate of potash by dissolving uric acid in KOH with the aid of
114 Practical Physiology.
gentle heat. Allow it to stand for forty-eiglit hours — the urate
deposit which results then behaves like the urates found in urine
i.e., disappears on heating and reappears on cooling.
(259) Test a small amount of the concentrated urate of
potash for reduction with Fehling's solution A whitish pre-
cipitate of cuprous urate forms which on further heating gives
some reduction. Reduction is also shown by Schiff's Test: —
Dissolve some uric acid in sodium carbonate solution, moisten a
filter-paper with it, apply a drop of silver nitrate, a black stain
of reduced silver appears. In human urine the concentration
of urates is insufficient to give reduction with Fehling's
solution.
(260) Dissolve some uric acid in ammonia solution, add
magnesia mixture and a few drops of silver nitrate solution. A
gelatinous precipitate of silver-magnesium-urate forms.
(261) Murexide Test. — This is the most delicate test for
uric acid, but it is given in modified form by the xanthin bases
and a similar colour is produced with cholesterin (77).
To a few particles of uric acid or to several drops of urine
add about five drops of nitric acid ; evaporate gently on a small
flame ; when dry, cool and add ammonia at one point — a purple
colour results. The colour is due to the formation of ammonium
purpurate — purpuric acid having resulted from the oxidation
of the uric acid. If KOH is added to the residue at another
point — a bluish violet colour is obtained.
(262) Xanthin Bases. — These occur in small amounts along
with uric acid. To about 20 c.c. urine add 5 c.c. magnesia
mixture — a precipitate of phosphates results. Filter and add
2 — 4 c.c. silver nitrate solution to the filtrate. If the precipitate
which results is white and curdy add more ammonia till the
white (AgCl) disappears. Allow to stand for a little — a fine
gelatinous precipitate settles out, consisting of a compound of
silver and magnesium, with all the purin bodies. (There is a
clinical method (Walker- Hall's) for estimating purin bodies,
according to the amount of this precipitate, in the same way as
albumin is estimated by Esbach's tube).
Peaotical Physiology. 115
The term "alloxuric bodies" or "purin bodies" is applied
to the group which includes uric acid and the nuclein or xanthin
bases (xanthin, hypoxanthin, guanine, and adenin).
(263) Creatinine. — Repeat WeyVs and Jaffa's tests (125),
(126), using normal urine instead of the muscle extract.
Creatinine in strong solution gives reduction tests, but it
has also the power of holding reduced oxide of copper in solution,
and so prevents the reduction from becoming apparent to the
eye. It is therefore an important factor in obscuring the
reduction test in urine where the amount of the glucose is very
small (MacLean).
(264) Hippuric Acid. — (Always present in herbivorous
urine — occurs in variable amount in man). Heat a small quan-
tity of hippuric acid in a dry test tube. The crystals melt —
become dark red — and give off an aromatic odour like that of
oil of bitter almonds, due to phen^dcyanide and hydrocyanic
acid.
(265) Organic or Ethereal Sulphates. — These are bi-
sulphates of potassium and sodium in which the hydrogen is
replaced by indoxyl, skatoxyl, phenol, etc. The chief of these is
indican or indoxyl-sulphate of potassium.
(a) To about ten c.o. of herbivorous urine add an equal
amount of concentrated HCl, several drops of chloroform, and
one drop of a 5% solution of commercial chloride of lime, or
"bleaching powder ;" shake ; the indoxyl is set free by the HCl,
oxidised to indigo-blue by the hypochlorite present in the bleaching
powder, and this is dissolved and carried down by the chloroform
which forms a blue-coloured layer at the bottom of the tube
when the result is positive. A second drop or more of the
bleaching powder solution may be added if the bine colour does
not appear at first, but excess of this reagent must be avoided
as it may oxidise the indigo-blue to a colourless compound. If
human urine gives the test, the indication is that there is an
abnormal amount of bacterial action going on in the alimentary
tract.
116 Practical Physiology.
(b) The sul-phate part of the ethereal sulphate may be tested
for as follows : — Add to the urine some acetic acid, and then barium
chloride solution in excess ; keep the mixture on the water-
bath till the precipitate settles ; filter or decant off the clear
supernatant fluid ; divide into two parts ; to one add some
HCl and boil ; compare it with the other for the presence of a
precipitate (disregard any colour changes which may result on
boiling with HCl). The rationale of the test is this : — The in-
organic sulphates are precipitated by the barium chloride first
added, the phosphates being held in solution by the acetic acid ;
the filtrate contains the excess of barium chloride and the ethereal
sulphates ; when this is boiled with HCl these are broken up,
and yield inorganic sulphate, which unites with the barium present
to give a further, though much smaller, precipitate of BaS04.
(266) Total Nitrogen. — The nitrogen of the urine is chiefly
contained in the foregoing substances — urea, uric acid, xanthin
bases, creatinine, hippuric acid, and indican ; a variable amount
also exists as ammonium salts and in the pigments, mucin, etc.
The total amount rnay be estimated by Kjeldahl's method as
follows : — Measure accurately 5 c.c. of urine into a round-
bottomed Jena glass flask, specially used for these estimations ;
add lOc.c. of pure H2SO4, a crystal of CuSO.j, and a few grammes
(5-10) of potassium sulphate. Heat the flask over a bunsen
flame on a tripod with wire gauze in a fume chamber ; the flask
should be supported in an inclined position. Under the influence
of the high temperature and acid the whole of the nitrogen becomes
converted into ammonium sulphate, and the other elements are
oxidised, e.q., carbon forms CO.,, hydrogen HgO, etc. Allow the
incineration to proceed for some time after the contents become
clear, then cool and add water to dissolve the residue. The
ammonia in this residue is then distilled over after adding strong
NaOH, and estimated as described later (Exp. 306, C.3.).
(B) INORGANIC CONSTITUENTS.
Chlorides. — Test as in (2), acidify with nitric acid to prevent
phosphates from being precipitated, and add silver nitrate.
Practical Physiology. 117
(267) Quantitative Estimation of Chlorides (Volhard). —
Measure accurately 10 c.c. urine into a 100 c.c. flask, add some
distilled water, 4 c.c. of pure nitric acid, and 15 c.c. of standard
silver nitrate solution measured from a burette, then fill up to
the 100 C.c. mark with distilled water and shake. This causes
precipitation of all the chlorides, and leaves some silver nitrate
in solution, the amount of which is now to be estimated. Filter
the contents of the flask through a dry funnel and paper into a
dry 50 c.c. flask (or if the flask is wet it may be washed out with
the portion of the filtrate which first runs through), Collect
exactly 50 c.c. of the filtrate, empty it into a titration flask or
beaker, and wash out the flask, and add the washings to the 50 c.c.
Then add about 5 c.c. of ammonia iron alum solution to act as
an indicator, and run in carefully from a burette sufficient standard
ammonium sulpho-cyanide to cause a permanent red tinge in the
fluid. Note the starting and ending points ; calculation —
Suppose 15 c.c. of AgNOg were taken (more may be required if
the urine is rich in chlorides), and that 3 c.c. of NH4CNS solu-
tion were sufficient to produce the end reaction with half the
filtrate {i.e., 50 c.c), then 6 c.c. would have been required if the
whole filtrate had been taken. Since 1 c.c. of sulpho-cyanide
exactly combines with 1 c.c. of silver nitrate, the excess of silver
nitrate in solution in the 100 c.c. flask after precipitating the
chlorides is 6 c.c, and therefore 9 c.c. must have combined with
the chlorides of the urine. Now 1 c.c. of the standard sUver
nitrate solution corresponds to 0.01 grm. of sodium chloride, and
therefore the 10 c.c. of urine contains 0.09 grm. of chlorides
reckoned as NaCl = 0.9% or 13.5 grm. in 1,500 c.c
The strength of the standard silver nitrate is obtained from
the combining weights of AgNOg and NaCl, thus NaCl (58.5 grm)
-j- AgNOg (170 grm) exactly satisfy each other, and therefore
29.06 grm. AgNOs in a litre of water would equal 10 grm. NaCl
or 1 c.c. of the silver nitrate solution would equal 10 mlgm. of
NaCl. The ammonia iron alum acts as an indicator in this way,
that as soon as sulpho-cyanide has been added in amount sufficient
to combine with the silver present, the next drop strikes a red
colour with the iron salt ; cf. (160) and (213).
(268) Phosphates. — Simply heating the urine causes pre-
cipitation of earthy (Ca and Mg) phosphates in some cases by
118 Practical Physiology.
driving off the CO, ; the precipitate is soluble in acetic acid
(distinction from albumin, see test (21) ). Addition of alkalis
or ammonia causes precipitation of the same varieties of phos-
phate ; filter : the filtrate still gives the tests for phosphates (8)
and (4) due to the presence of alkaline phosphates (K and Na).
Traces of organic phosphates occur corresponding to the ethereal
sulphates.
(269) Estimation of Phosphates (Uranium Nitrate Method).
Measure accurately 50 c.c. urine into a small porcelain basin, add
about 5 c.c. of sodium acetate solution and heat to boiling.
Have ready a burette containing uranium nitrate, and a series of
drops of potassium ferrocyanide arranged on a white porcelain
slab. When the urine begins to boil, gradually run in some
uranium nitrate solution from the burette, stir with a glass rod
and test a small drop of the boOing mixture for free uranium
nitrate by bringing it in contact with one of the drops of ferro-
cyanide. Cease adding the uranium nitrate when the mixture,
tested in this way, shows a faint brown tinge, which indicates that
all the phosphate in the urine has been precipitated as phosp hate
of uranium and that any further addition of uranium nitrate
remains free in solution. Read the amount of uranium solution
used to reach this end point ; it is so made that 1 c.c.of it equals
5 mlgm. (.005 grm.) of PgO,. From this it is easy to calculate
the amount in percentage, or as total daily output if the amount of
urine is given.
The reason for adding sodium acetate is that during the action
between the acid phosphates of the urine and the uranium nitrate
some free nitric acid is liberated ; this would keep the uranium
phosphate in solution if it were not for the sodium acetate which
combines with the nitric acid and sets free acetic acid instead.
Acetic acid is also added to the sodium acetate solution when it
is made up ; it keeps the earthy phosphates in solution till they
combine with the uranium.
(270) Sulphates (inorganic) see (265 6) and (6) ; about one-
tenth of the sulphate present in urine is in organic combination.
(271) The hases united to the foregoing acid radicles or free
as kations, are sodium (7), potassium (8), calcium (9), magnesium
Practical Physiology. 119
(10) and ammonia. The best way to demonstrate the presence
of calcium and magnesium is to add to about 100 c.c. of urine
sufficient ammonia to cause a copious precipitate of the earthy
phosphates ; filter ; dissolve the precipitate from the filter paper
with dilute acetic acid. This gives a concentration of the Ca and
Mg salts, and on adding ammonium oxalate a large precipitate
of calcium oxalate comes down ; filter till clear, and add an excess
of strong ammonia to the filtrate — the magnesium comes down
as ammonio-magnesium phosphate.
Ammonia may be detected and estimated as follows : —
(Schlosing's Method). A bell-jar fitting accurately on a glass
plate is required ; place 20 c.c. of ^ acid in a small beaker or
porcelain basin ; over this, on a tripod, place another basin
containing 20 c.c. of fresh urine with thymol added to prevent
fermentation ; add to this latter about the same amount of
' ' milk of lime ' ' ; these are all arranged on the glass plate and
then covered with the bell-jar, and the whole made air-tight by
smearing the junction of the bell-jar and plate with vaseline. Let
this stand for three days ; then estimate the diminution of acidity
of the -Yo acid by titrating with y'^ alkali. The ammonia has been
gradually liberated by the lime and is then absorbed by the acid.
On boiling some urine with KOH there is, of course, a copious
evolution ot ammonia derived from the urea, nitric acid, etc., but
lime at room temperature does not attack these bodies.
(271a) Deposits in Urine. — The common forms of deposit
in urine are : — (1) A pinkish coloured deposit of urates consisting
of amorphous granules ; it occurs in acid urine but persists if
the urine becomes alkaline from decomposition. Urates dissolve
on gently heating (256). (2) Uric acid — fine dark scattered
grains like cayenne pepper — of characteristic form under the
microscope. (3) Calcium oxalate, less easily distinguished macro-
scopically because always small in amount. The crystals are
clear, colourless, octahedra as a rule, occasionally dumb-bell
shaped. (4) A white deposit in an alkaline urine is generally
ammonio-magnesium fhosphate. The crystals have the form
of " knife 'rests," or "feathery" phosphates. (5) "Stellar
phosphates ' ' or fhosfhate of calcium has a characteristic appear-
ance under the microscope. (6) Pus can be distinguished by the
120 Practical Physiology.
appearance of pus corpuscles when examined under the micro-
scope ; the deposit becomes glairy on addition of KOH, and
forms " strings " on pouring it from one test tube to another ;
pus in urine gives the guaiac reaction. The rare forms of urinary
deposits are, leucin usually associated with tyrosin, cystin, hippuric
acid, etc.
If the suspended matter present in the urine is sufficient to
form a distinct deposit, one can easily obtain some of it for
microscopic examination, but when the urine is suspected of
having material present which does not form a sediment one may
use a small centrifuge and obtain a drop from the narrow end of
the centrifuge tube for examination. In this way hlood cells,
epithelium from the Icidney and urinary passages, tvhe casts, bacteria.
Ho., may be found. (Demonstration on the use of the small
centrifuge).
Normal urine may frequently show a fine cloud of mucus
as a deposit. It consists of mucin precipitated by the acidity
of the urine. The mucin is not a true urinary constituent, but
is added to it by the epithelium of the urinary passages.
(G) PATHOLOGICAL CONSTITUENTS OF URINE.
(272) Albumin. — This is serum albumin usually accompanied
by serum globulin. The tests commonly employed in clinical
work are {a) coagulation on heating, see test (21) ; (6) Heller's test,
nitric acid in the cold by the ring method (22) ; (c) acetic acid and
potassium ferrocyanide (23) ; {d) Eshach's solution used for estim-
ating albumin may also be used qualitatively. In every case
the urine ought to be clear before the tests are applied, therefore
filter if necessary ; and more than one test should be employed.
The amount of albumin may be approximately determined
with Esbach's Albuminimeter as follows : — Clear the urine by
filtering if necessary ; take the specific gravity, and if it is over
1010 dilute the urine with a known amount of water till it is at
or under that figure, e.g., if the specific gravity is 1015 take 30 c.c.
dilute up to 45 c.c, and mix thoroughly ; fill the Esbach tube with
Practical Physiology. 121
urine up to the mark U ; add the reagent up to mark R ; cork
the tube, mix, let stand for one day, and read the height of the
precipitate. In the above case the result would require to be
increased in the ratio of 30 : 45 to give the true figure. (See also
156). The urine may require dilution not only on account of
a high specific gravity but also because of an excessive amount of
albumin. The urine in albuminuria may be pale and of low spec-
ific gravity, or concentrated and contain blood, or glucose or
bile may also be present.
(273) Blood. — Red and white corpuscles may occur, or
haemoglobin, or one of its derivatives such as methaemoglobin
may be present without the corpuscular elements.
The urine may appear reddish, but if there is only a small
quantity of blood it has a characteristic ' ' smoky ' ' appearance.
(a) If blood is present the Guaiac Test (159) should give a
positive result, but this is not an infallible indication of its pres-
ence ; the reagents must be active — test with a few drops of
blood from the finger mixed with water ; and some drugs when
excreted in the urine give a similar reaction. Such drugs are
rhubarb, senna, santonin and iodides. Oil of turpentine, which
has been long exposed to the light and which then contains ozone
may be used instead of ozonic ether.
(b) Spectroscopic examination of the urine (161) is the best.
The spectrum of HbO^, or of one of its derivatives may be seen
directly in the urine, or these pigments may be converted into
haemochromogen (166) which in dilute solution has a more intense
spectrum than any of the others. The blood pigment may be
concentrated from a large bulk of urine, say 100 c.c, by forming
a precipitate in it of the earthy phosphates with KOH, or of
albumin coagulated by heat ; the haemoglobin or its derivative
goes down along with the precipitate or coagulum, and can then be
extracted with a small amount of dilute II2SO4 ; then render
alkaline, heat, cool, add ammon. sulphide and examine for
haemochromogen (166).
(c) Microscopic examination of the deposit from the urine,
or of the result of centrifuging may show the presence of blood
cells.
122 Practical Physiology.
(d) Heller's Test for Blood. — Take about half full of a test
tube of the urine, make it strongly alkaline with caustic alkali,
boil ; if a moderate amount of blood is present the deposit of
earthy phosphates which is thrown down is coloured brownish-
red by the haematin formed, while the supernatant fluid has a
greenish tint.
(274) Bile. Bile Pigment, (a) Apply Gmelin's Test (235)
directly to the urine. In the play of colour look especially for
the green (biliverdin) because non-bilious urine may give some
colour with the nitric acid due to indican and skatoxyl. (b) A
more certain, though slower, method is to filter a large amount
of the urine repeatedly through the same filter paper ; the paper
takes up the pigment ; then spread the paper out flat on a porce-
lain slab and place a drop of the impure nitric acid on the centre ;
the rings of colour appear around the spot, (c) Huppert's modi-
fication of the test is : — add a small amount of milk of lime to
about 50 c.c. of the urine ; this carries down the pigment ; filter
of[ the sediment ; wash with water on the filter paper ; dissolve
in acid alcohol (5 c.c. of HCl in 100 c.c. methylated spirit) ; heat
the alcoholic solution — if bile pigment is present the solution
becomes green or bluish green.
Bile Salts. — These occur less frequently in urine.
(a) Try Pettenlcofer's Test with spectroscopic examination of
the colour '(234) ; (b) the sulphur test (236) ; (c) Oli"er's Tes'—
Acidify slightly with acetic acid and filter if the urine is alkaline
or turbid, or both ; reduce the specific gravity to 1008 if necessary,
add to the urine three times its bulk of a peptone solution — a
milky opalescence indicates bile salts.
The peptone solution consists of powdered peptone (Savory and
Moore's is best), half a drachm ; salicylic acid, 4 grains ;
acetic acid, half a drachm ; distilled water, up to 8 ounces ;
filter till clear.
(275) Sugar (Glucose).— Diabetic urine in a case of even
moderate severity is clear, and of high specific gravity (above 1030)
and large amounts are passed per day.
Practical Physiology. 123
(a) It gives all the ordinary reduction tests.
(b) It gives the phenyl glncosazone crystals.
(c) It ferments easily.
(d) And rotates the plane of polarised light.
Repeat tests (42) to (51).
(276) Estimation of Glucose. — Glucose may be estimated by
the polarimeter (or saccharimeter) or by its power of reducing
cupric salts.
(a) The ordinary Fehlinq Method. — Fehling's solution, when
freshly made, is of such a strength that each c.c. is completely
reduced by 5 mlgm. (.005 grm.) glucose. The urine generally
requires dilution from 1 to 20 times with water. This must be
done by measuring accurately 10 c.c. of urine from a graduated
pipette or burette, and making it up to 100 c.c. with water. Place
the diluted urine in a burette. Measure accurately 10 c.c.
Fehling solution into a porcelain basin, add some water and boil.
While the fluid is kept boiling run in the diluted urine, stirring all
the time, till the last trace of the blue colour of the Fehling's
solution has gone. The colour may reappear when the fluid is
allowed to cool in air, but this is of no consequence. During the
estimation the fluid must be kept boiling. The end reaction is
somewhat difficult to preceive ; a good plan is to continue the
boihng for a little time till the deposit forms then tilt the basin
slightly, and look through the edge of the hot fluid. Read the
amount of diluted urine necessary to reduce the 10 c.c. Fehling =
X c.c, then X c.c. contain .05 grm. glucose. If the dilution is 1
in 20, then ^ of the original urine contains .05 grm. glucose, and
20 X. 05x100 ,,
=z = the percentage.
X
A rough indication of the amount of glucose present in
urine may be done as follows : To. freshly prepared Fehling in
a test tube add an equal amount of the urine ; boil for about a
minute. If all the blue colour disappears the urine contains
.05% or more glucose. If after boiling 1 volume of urine with
2 volumes of Fehling, the reduction is complete, then 1% or more
present. In the same way complete reduction in a mixture of
1 of urine with 4 of Fehling indicates 2% or more.
124 Practical Physiology.
(b) Pavy's Modification of Fehling's Method (Dem.) gives a
sharper end reaction. The reduction is done in a flask closed except
for a steam outlet and the nozzle of the burette, both of which
pierce the stopper. The flask is suspended over the bunsen flame
by means of the rubber tubing'which connects the burette to it?
nozzle. The copper solution used contains an excess of NH3
which holds the reduced copper oxide in solution (see 43), and so
one can tell more readily when the solution is fully reduced, i.e.;
when all the blue colour is gone. The procedure is as follows :
50 c.c. of Pavy-Fehling solution is placed in the flask. This is
equivalent to .025 grm. glucose, for the Pavy-Fehling contains
10 times less copper than ordinary Fehling. Place the diluted
urine in the burette, see that the nozzle is full to the point, and
then insert the stopper into the flask, and proceed to boil the con-
tents. The best dilution of urine to employ depends on the amount
present. It should be such that the diluted solution has about
0.1% glucose (and this can be determined roughly in a pre-
liminary test).
When steam begins to issue freely from the flask run in the
diluted urine gradually, having first read the level of the fluid
in the burette. When sufficient glucose has been run in, the blue
colour is completely discharged, and this constitutes the end point.
The calculation is easily understood. 50 c.c. Pavy-Fehling fully
reduced means .025 grm. glucose in the amount of diluted urine
run in ; say X c.c, and suppose the original urine has been diluted
1 in M parts, then ^c.c. contains .025 grm. glucose, and 100 c.c.
... ^ . 100 X M X .025 . , , ,,,,,,
will contain „ = %. A great deal 01 the accuracy
in this method depends on the rate of delivery of the urine from
the burette. If too slow the NH3 boils off before the end point
is reached, and a deposit of cuprous oxide comes down which
spoils the reaction. If the delivery is too rapid, one is apt to get
beyond the end point because the reduction does not occur im-
mediately. The entrance of air into the flask leads to re-oxidation
of reduced copper, consequently the flask must not be allowed
to cool, nor must one add too much cold diluted urine at a time.
(c) The saccharimeter may be used (51).
Practical Physiology. 125
The reactions generally designated tests for '" sugar " (Trommer.
Fehling, Moore, etc) inerely indicate the presence of a
reducing substance in the urine. The results of these tests
should be confirmed by the application of the Phenyl-
hydrazin test, fermentation, or the polarimeter.
In the great majority of cases glucose is the reducing substance,
but occasionally the following causes of reduction are met
with (277) to (279).
(277) Lactose (See under Disaccharides c). — Lactose appears
occasionally in the urine of nursing women. It gives the reduc-
tion tests except Barfoed, but t^is latter can hardly be depended
upon as a crucial test. More important is the negative result
with yeast fermentation witliin 12 to 24 hours. If left longer
inversion of the lactose may occur, and then fermentation. The
crystals of phenyl-lactosazone are somewhat characteristic in
appearance, — the clusters of needles being more ball-like than
those of glucose.
(278) Pentose. — This monosaccharide (G^JL^gO^) may occur
in urine after eating certain fruits, such as pears, or as a rare disease
condition (pentosuria) ; probably it frequently occurs undetected
along with glucose iu diabetic urine. It gives positive results
with the reduction tests, yields an osazone with a low melting point
(158° C), does not ferment with yeast, gives a red colour and a
red precipitate when warmed with an equal amount of concen-
trated HCl and a few grains of dry fhloroglucin ; the orcin test
is carried out in the same way with orcin instead of fhloroglucin ;
if pentose is present the fluid becomes reddish-blue, then bluish-
green, and shows a spectrum with an absorption band between
C and D. Do these tests on the pentose solution provided.
(279) Glycuronic Acid occurs in traces in normal urine, and
as a compound with such substances as morphine, chloral and
chloroform when these are excreted in the urine. It may be
present in sufficient amoiint to give reduction. It gives the
phloroglucin but not the orcin test as applied above ; it does not
ferment ; and it can be distinguished from other substances bv
a peculiarity in its optical activity — as it occurs in urine it is
Irovo-rotatory, but when boiled with acid and so dissociated from
its combinations the free glycuronic acid is dextro-rotatory.
126 Practical PHYSTOLoar.
It is important to note that the reducing substance in urine
which is of the greatest significance (glucose) is readily fermentable
with yeast ; and further, that urine contains a very small but
definite amount of reducing substances the chief of which is
glucose ; probably owing to the creatinine, however, normal
urine does not reduce Fehling, and gives no result on fermenta-
tion.
The Acetone Bodies (beta-hydroxybutyric acid, aceto-acetic
acid, and acetone). These may occur in the urine in diabetes
and to a less extent in some other conditions. Normal urine
contains traces of acetone, however. They are closely related
to each other and one may change into the other in the above
order. There is no convenient clinical test for the first.
(280) Aceto- Acetic Acid. — Add to the freshly passed urine
a few drops of ferric chloride — a precipitate of phosphate of iron
will appear, continue adding the ferric chloride till no more
precipitate comes down, filter and add a further small quantity of
ferric chloride to the filtrate — a reddish claret colour indicates
the presence of aceto-acetic acid. The claret colour may be
sufficiently evident in the urine before filtering. Salicylates,
if being taken by the patient, cause a similar reaction, but in such
a case the result is still present after boiling the urine.
(281) Acetone and Aceto-acetic acid together, w acetone alone
gives the following reactions : — (a) Iodoform test. Make the
urine alkaline with KOH, add a few drops of watery solution
of iodine in potassium iodide, a white turbidity and smell of
iodoform indicates the presence of acetone.
(b) Legal's Test. Add to the urine a few drops of freshly
prepared sodium nitroprusside solution and make alkaline with
KOH. The urine becomes of a ruby-red colour ; a similar colour
appear? in testing for creatinine (125) but a distinction can be
made between these two, in this, that the addition of acetic acid
in the case of creatinine causes the colour to change to yellow,
green, and then blue, while with an acetone solution the colour
becomes magenta on adding acetic acid. A recent modification
of the test (Rothera's) is as follows : — Add a little solid ammonium
Practical Physiology. 127
sulphate to 5-10 c.c. of the urine, then 2-3 drops of a fresh- 5%
solution of sodium nitroprusside, and 1-2 c.c. of strong ammonia,
a characteristic " permanganate " colour develops. In the
concentration in which it exists in urine creatinine gives no
result with the nitroprusside test applied in this way, and it is
moreover exceedingly delicate towards acetone.
Since the acetone bodies are volatile, they can be detected
by their fruity odour, and can be distilled off from a large quantity
of urine after acidifying with acetic acid ; the distillate must be
received into a flask kept as cool as possible, preferably with ice.
(282) Secondary albumose in urine ('Peptonuria "). Satu-
rate the urine at boiling temperature with ammonium sulphate
crvstals filter, wash the precipitate with hot saturated ammonium
sulphate solution, extract the filter paper and its contents with
hot water, filter again, and do the biuret test (17). Urobilin may
complicate the result in a highly coloured urine.
(For an account of the normal and pathological pigments
of urine consult Milroy's Practical Physiological Chemistry).
(283) Cryoscopy or Depression of the Freezing Point. The
apparatus required consists of a large jar to hold the freezing
mixture which may consist of alternate layers of crushed ice and
common salt ; the lid of the jar is of wood and carries a large test
tube within which, supported by a ring of cork is a smaller test
tube to hold the fluid experimented on, {e.g., urine) ; a special
thermometer with a scale divided to read to hundredth's of a
degree C. is also placed in the inner tube, along with a simple spiral
wire of platinum or copper to -act as a stirrer.
Arrange the apparatus as above, and watch the temperature
as it slowly falls ; when it reaches the freezing point of pure
water, stir vigorously and keep stirring till the reaction is ended ;
the temperature sinks to a variable extent below the actual freez-
ing point of the fluid, and when ice begins to form it suddenly rises
and if left in contact with the freezing mixture it again slowly
falls ; the point to which it suddenly rises before beginning to
128 Practical Physiology.
fall the second time is the freezing point of the fluid (urine). If
the thermometer employed is graduated in the ordinary way
with 0° as the freezing point, the depression of freezing point can
be directly read, but where a Beckmaim thermometer with an
adjustable zero is used the freezing point of pure distilled water
must be determined. The difference is then the depression of
freezing point.
The average depression for urine is 1.85° C.
CHAPTER X.
METABOLISM AND DIETETICS.
(284) Storage of Glycogen in the Liver.
The presence of a large amount of glycogen can be demon-
strated in the liver of an animal fed on carbohydrate-rich food
some hours before being killed. Rapidly excise the liver just
after death ; keep one small bit separate and rapidly cut up the
remainder in boiling acidified water ; after the pieces have become
coagulated remove them from the fluid, grind them up in a mortar
with fine clean sand, and return the pulpy mass to the water ;
continue the boiling for ten to fifteen minutes ; filter and dis-
tribute the filtrate to the class. Cut up the small separate piece
of liver in a beaker of normal saline, place it in the incubator,
and examine it next day for glycogen and glucose.
Further treatment of the filtrate. — First remove any protein
that may be left by adding alternately a drop or two of HCl and
of potassio-mercuric iodide (Briicke's Reagent) till no further
precipitate of protein appears ; then filter ; measure the filtrate
and add to it twice its volume of alcohol to precipitate the gly-
cogen ; let the precipitate form and settle till next day then filter
and wash with 66% alcohol ; then add some boiling water to the
precipitate on the filter paper ; the glycogen goes into solution
and comes through in the filtrate. Test this glycogen solution
as in tests (67) to (71) inclusive. Test also the efiect of saliva
(ptyalin) on it (214).
130 Practical Physiology.
In the separate piece of liver which was kept as 40° C.
practically all the glycogen will be found to have been converted
into glucose.
(285) Phloridzin Glycosuria. — This experiment is to be carried
out by two members of the class chosen by lot. A rabbit is placed
in a collecting cage, fed on the usual diet, and normal urine
collected for a day. Then inject subcutaneously about quarter
of a gramme of phloridzin dissolved in warm water. Continue to
collect the urine in a fresh receptacle or catheterize the animal
after several hours. Samples of the normal urine and of the
phloridzin urine are to be tested for glucose by the class as a whole.
QUANTITATIVE ANALYSIS OF SOME FOOD STUFFS.
Some of these have been already examined, e.g., meat (muscle),
meat extract, gelatine, starch, cane sugar, and fats.
Milk. — The chief solid constituents found in all kinds of milk
are proteins, fats, and carbohydrates, and salts in varying pro-
portions to suit the environment and rate of growth of the young
mammal.
The proteins are easeinogen (a phospho-protein), and small
amounts of lact-albumin and lacto-globulin ; the fats are tripal-
mitin, tristearin, triolein with glycerides of some of the lower and
volatile fatty acids, e.g., tributyrin ; the carbohydrate is lactose ;
the salts are fotassium, calcium, sodium, magnesium as phosphates,
chlorides and sulphates. Cow's milk is put out for examination.
(286) The reaction to litmus paper is alkaline, or amphoteric
(i.e , changing blue litmus red, and red litmus blue) in the fresh
state, but it readily becomes acid on standing, due to the action
of bacteria (B. lactis, etc.).
(287) The specific gravity varies from 1028 to 1034 and is
higher when fat has been extracted, lower when water is added.
(288) Microscopically it shows fine fat globules of varying
size.
(289) Milk gives the Guaiac Reaction (159).
Practical Physiology. 131
(290) Separation of the chief constituents of milli. To about
10 c.c. of milk in a large test tube add four to five times its bulk
of water and acidify very cautiously with acetic acid, mixing
thoroughly after the addition of each drop (two drops may prove
sufficient) — a flocculent precipitate of caseinogen and entangled
fat appears. This precipitate readily dissolves in excess of the
acid as can be shown by pouring some of the fluid into another test
tube and adding more acetic acid. Filter, and keep the filtrate ;
wash the precipitate on the filter paper with water containing
a trace of acetic acid (discard the washings) ; dissolve some of
the precipitate in very dilute sodium carbonate solution and
apply some of the protein colour reactions (15--20). Caseinogen
is rich in aromatic substances (tyrosin, tryptophane, etc.) and
therefore gives positive results with (15), (16), (18) ; as it contains
no carbohydrate radicle tests (19) and (20) are negative. Allow
the remainder of the precipitate of caseinogen -j- fat to dry in the
oven till next day then extract some of the fat with ether, note
the yellow colour of its ethereal solution and the greasy stain
left on note paper where the ethereal solution has been allowed
to dry (see 293) ; incinerate the remainder of the precipitate as
in (74) and test for phosphate (3) (4).
The filtrate from the caseinogen-and-fat precipitate contains
the other constituents. Test it for lact-alhumin and lacto-qlobulin
together by boiling after adding a little common salt (see 21.)
Filter off the coagula of these and test the filtrate for lactose
(see under Di-saccharides, C) ; test another portion of the filtrate
for calcium (9) and for inorganic phosfhates (3) (4).
(291) The action of rennet on milk can be again studied (226).
Compare the amount of lime present in the whey with the amount
present in a filtrate after precipitating the caseinogen as above,
after diluting the whey to the same extent as the milk was diluted.
The curd which results from rennet coagulation consists partlv
of the lime of the milk, and whey is therefore relatively poor
in that element.
Cheese consists chiefly of casein and fat, part of which arises
from the casein itself. The lactose originally present in the milk
forms only a small percentage of the solids. Among the salts
present are calcium and sodium, as phosphates and chlorides.
132 Practical Physiology.
(292) Rub up some finely grated cheese with 2% sodium
carbonate in a mortar and filter. The casein is to some extent
dissolved and gives reactions very like those of caseinogen, e.g.,
it is precipitated by acetic acid and the precipitate is soluble in
excess. It gives the protein reactions, especially Millon's and
xanthoproteic, (aromatic radicle).
(29-3) Fat. — Extract some grated cheese in a dry test tub^
with ether, pour the ethereal solution into a warm porcelain basin,
and allow it to evaporate to small bulk, then place a drop of the
fluid on clean white note paper, and allow the last of the ether to
evaporate — a greasy translucent spot remains indicating the
presence of fat.
The fat can also be detected by saponification, but should be
first extracted in larger quantity by ether. The salts may be
examined after incineration in the usual way.
Eggs (that of the hen is used for convenience). The white
of egg consists chiefly of water with about 12% of proteins (egg-
albumin, ovoglobulin, and ovomucoid). The yolk contains about
32% of fatty matter soluble in ether (true fat, lecithin, lutein,
cholesterin, etc.) ; proteins constitute about 16%, the varieties
being chiefly vitellin (a phospho-protein) and nucleo-protein ;
water and salts are the chief other constituents of the yolk.
(Iron is present in egg-yolk in organic combination to the extent
of .01%. It is relatively much richer in iron than milk is).
The following constituents of egg-yolk may be examined.
(294) Fat and Lutein. — Extract some fresh egg-yolk with
ether by shaking in a test tube ; pour off the upper ethereal layer
and examine it as above described (293) for fat, and with the
spectroscope for the two bands of lutein seen near the violet end
of the spectrum ; one of these bands is over F, and one lies be-
tween F and G. Eepeat the extraction of the yolk with several
fresh quantities of ether so as to free it from as much fatty matter
as possible. Keep the ethereal extracts for examination of the
lecithin and for recovery of the ether.
Practical Physiology. 133
(295) Yitellin — The fresh egg-yolk usad above, which has
had its fatty constituents removed by ether, is now to be placed in
a porcelain capsule on a warm bath for a short time to remove the
ether ; then place in a large test tube, add 10 % sodium chloride
to nearly fill the tube, shake thoroughly and leave standing till
next day. Then filter and pour the filtrate into a jar full of
distilled water to which a few drops of acetic acid have been added;
a precipitate of vitellin will settle out in fine flakes ; after standing
some hours pour off the supernatant fluid and dissolve the pre-
cipitate of vitellin in the least necessary amount of weak sodium
carbonate solution. On this solution of relatively pure vitellin
do the protein colour reactions (15-20) especially those for the
aromatic radicle. Neutralise carefully the remainder of the
alkaline solution of vitellin, add an equal amount of 0.4% HCl
to make the precentage of the mixture 0.2, add an active pepsin
preparation and allow the mixture to digest till next day ; a
sediment will then be found consisting chiefly of paranuclein
derived from the vitellin. If filtered off, dried and incinerated
with NagCOg this deposit will be found to be very rich in phos-
phorus. (Compare carefully the reactions of vitellin with those
of caseinogen ; both are phospho-proteins, rich in aromatic
radicles and destined for use in building up the body of the young
animal ; they resemble nucleo-proteins in some respects, but that
there is some great difference in the constitution of the two
groups is shown by the fact that nucleo-proteins yield the
xanthin bases on decomposition while phospho-proteins do not.)
(296) The Mineral Constituents. Incinerate a small piece
of dried yolk as described in (1) and examine for the usual con-
stituents of a tissue (2) to (10). Note the relative amounts of
these as far as can be judged by qualitative tests ; since the yolk
contains practically all the constituents of the formed chick,
the amounts of the mineral salts found represent an average in
each case of all the tissues taken together. Make a final ex-
traction of the incineration residue with 1 in 5 HCl and test for
iron with a few drops of ammonium sulphocyanide (blood red
colour) or with potassium ferrocyanide (Prussian Blue).
(297) The Substances Soluble in Ether have already been
mentioned. After distiUing ofi the ether and obtaining an ether-
134 Peactical Physiology.
free residue a series of operations may be carried out to separate
the various substances. If time permits this may be carried out
as given in Milroy's Practical Physiological Chemistry.
EXAMPLES OF SOME VEGETABLE FOODS.
These contain a preponderating amount of carbohydrates
(chiefly starches) but proteins are also present, sometimes in large
amount, and fats also occur, e.g., olive oil.
(298) Flour. Add a small quantity of water to some flour
in a porcelain basin, and knead it between the fingers till it forms
a tough dough, this is due to the gluten and other proteins be-
coming viscid, and entangling the starch grains ; wrap the dough
in a piece of muslin and work it about in a quantity of water in
the porcelain basin. The starch grains pass out into the water
Keep some of this fluid and test it later on for starch. Continue
washing the dough till no more starch comes out, then open up
the muslin and examine the gluten which remains. Suspend a
little of it in water and apply the tests for protein ; test the
washings of the dough, after boiling and cooling, for starch
(58-62), and for glucose (43). In good flour the latter should be
absent.
(299) Bread. — In bread-making, a dough essentially similar
to the above is first made, but yeast is added and fermentation
is allowed to proceed ; the development of the gas throughout
the dough renders it spongy, and the digestive juices can readily
penetrate. The baking is done at a high temperature, and a
certain amount of dextrin is formed in the crust.
Test bread for protein by applying a few drops of Millon's
reagent and allowing it to remain for a short time at room temper-
ature — a deep red stain results. The xanthoproteic test may
be similarly applied.
Make a watery extract of the "crumb" of bread, filter and
test for glucose, starch and protein. Make a similar watery
extract of the "crust", filter and test for dextrin; since the
extract contains starch and amidulin, it is necessary to preci-
pitate these by saturating with magnesium sulphate ; filter and
test the filtrate with iodine, cf. (70).
Peactical Physiology. 135
The following other food stuffs may be examined in a
similar way if time permits.
(300) Oatmeal contains little or no gluten, and hence does
not form a dough or bread in the same way as flour. It contains
10% of fatty matter. Examine dried oatmeal for fat, protein,
carbohydrate and mineral matter including iron.
(301) Pulses form an important source of protein in vege-
tarian diets, the percentage being as high as that of meat (20%).
Examine some pease-meal or lentil-meal in the same way.
(302) Potatoes, Carrots, Parsnips, Onions, etc., consist
chiefly of carbohydrates and water, with less than 2% of protein,
and less than 1% of fat, and varying amounts of cellulose fibre,
mineral matter, and extractives.
(303) In Fruits, such as grapes, pears, apples, etc., the chief
constituent besides water is carbohydrate, chiefly as sugars
(pentosanes in pears), along with varying amounts of organic
acids such as citric, etc. The " jelly" of many fruits used in
making jellies and preserves is a form of gum, not animal gelatine.
(304) Nuts contain a large amount of oil and protein, and
lesser amounts of carbohydrate than the above, but they are
less digestible than fruit or vegetables.
EXPERIMENT ON GENERAL METABOLISM.
(305) The following experiment illustrates the principles
of dietetics as well as the method of studying general meta-
bolism. The work is to be divided into separate parts. A, B,
C, etc, one of which is to be done by one or more students according
to the size of the practical class.
An omnivorous animal, such as a dog or rat is to be kept
on a fixed diet of which the protein, carbohydrate and fat is to
be estimated, and the intake and output of nitrogen, phosphorus,
chlorine, calcium or other element determined in the food and
excreta.
136 Peactical Physiology.
For class work rats are very convenient as they can be kept
in a simple collecting cage such as that shown in fig. 15. The
disadvantage of using a rat is that the faeces and urine are
obtained together, and the percentages of food constituents
absorbed cannot be stated, so that where a dog is available it
should be used in preference to rats.
For either kind of animal a suitable diet consists in oatmeal
and dried protein, and it can be prepared by adding a measured
amount of boiling water to the weighed-out quantities of meal
and protein.
Assuming that the experiment is continued for two to three
days in a dog, or four days (two periods of two days each) in the
case of the rats, the work involved will take the working time of
one week, about eight hours, in a class of eighteen working in
couples.
At the end of the experiment the lecturer will collect all
the figures and draw up a statement of the results which will
include — (1) the daily diet of the animal in terms of protein,
carbohydrate and fat ; (2) the energy value of the diet reckoned
from the amounts of the substances and controlled by a direct
combustion of the food in the bomb calorimeter ; (3) where the
urine and fseces are obtained separately the amount of each food
stuff absorbed can be calculated as a percentage of what is eaten ;
(4) the balance of the various elements, e.g., nitrogen, phosphorus,
calcium, etc. ; (5) the gain or loss of weight of the animal and
the probable nature of the loss, thus a negative balance of 1 grm.
nitrogen over the whole experiment might mean a loss of 6.25
grm. dried protein, or, since muscle contains 20% of protein,
31.25 grm. of "flesh." (6) If the heat value of the excreta
can be directly determined by the bomb calorimeter the
"balance of energy" can be stated. Thus, knowing that a
certain number of calories had been taken in as food, and
that the caloric value of the excreta was so much, the
difference represents what has been used by the body and
either expended in work and heat or partly stored as the
potential energy of glycogen and fat.
Practical Physiology.
137
Fig. 15.
Eat's cage for metabolism experiments.
138 Practical Physiology.
(A) THE WATER PERCENTAGE OF THE FOOD.
Apparently dry meal and dried protein, etc., contain a
certain amount of adherent moisture, the percentage of which
must be ascertained, as follows : — Procure a pair of well-fitting
watch-glasses with a suitable brass clip for holding them in
apposition. Clean, dry in the hot air oven, cool in the desi-
ccator, and weigh the glasses and clip on the delicate analytical
balance ; weigh out about two grammes of the material on an
ordinary balance, place this on one of the watch-glasses, cover
it with the other, put on the clip and weigh on the delicate balance
again. The difference in the exact weighings is the amount of
meal or other undried material taken. Undo the clip, remove
the one watch-glass and place all in the hot air oven at a tem-
perature of 100° — 120°C. After an hour or more at this tem-
perature, put on the upper watch-glass, clip both together, and
allow the apparatus to cool in the desiccator. When cold,
weigh again ; this last weight may be that of the dried material,
but in order to make sure that all the moisture has been driven
off, the drying, cooling and weighing must be repeated until the
whole attains a constant weight, or varies only by a milligramme
or thereby. The water lost in the drying can easily be obtained
by subtraction of the weight of the dried from that of the undried
material, and the result expressed in percentage. The esti-
mation must be done in duplicate, and if the results are divergent
by 0.5% or more, another couple of estimations should be done.
This method is not applicable to materials vfith. constituents
which are volatile.
(B) THE PERCENTAGE OF ASH.
This may be carried out on some of the same material as
was used for A. Transfer the contents of two of the pairs of
watch-glasses to a porcelain crucible which has previously been
weighed (after careful cleansing, drying in the oven and cooling
in the desiccator) ; see that every particle of the meal has been
transferred to the crucible. Or, about four or five grammes of
the undried material may be placed in the crucible, the weight
of which has just been determined, and the whole re-weighed.
Place the crucible on a clay triangle supported by a tripod as
in (1), and incinerate, using a small flame at first. When the
Practical Physiology. 139
mass is completely charred, cool, extract the mass several times
with small quantities of hot distilled water, filter the extract
each time through the same ash-free filter paper ; allow the
extracts to flow into a beaker and lay it aside at present. Then
place the filter paper and its contents in the crucible, dry the
whole in the oven and then contine the incineration till all
the carbon is completely gone (Fig. 1). Cool the crucible, place
it on the water bath and add to it some of the extract which was
made early in the process. Keep adding some of the extract
till it has all been evaporated to dryness in the crucible, including
the washings of the beaker : then clean the outside of the crucible
from any adherent matter, finish the drying in the hot air oven,
cool in the desiccator and weigh. Substract the weight of the
crucible, the difference is the amount of ash in a certain amount
of meal, etc. Express the result in percentage. The reason for
the preliminary extraction with water is that the chlorides are
to some extent driven off by the heat necessary to remove all
the carbon. It also facilitates the complete reduction of the
material to ash.
(C) THE PERCENTAGE OF PROTEIN.
Since it is difficult to separate the protein in a pure state
from meal, etc., the method usually employed is to estimate
the amount of nitrogen present, and multiply this by 6.25. This
is done on the assumption that protein is the only nitrogenous
substance present, and that the protein present is one that contains
16% of nitrogen ; it is therefore only approximately correct, but
is convenient for our purpose because we are about to determine
the total amount of nitrogen ingested and excreted. The nitrogen
is estimated by Kjeldahl method as follows : —
(1) Weighing the material. — About one gramme of oatmeal,
or half a gramme of a dried protein, such as casein, fibrin, etc.,
is weighed on an ordinary balance and transferred to a small dry
test tube. Prepare several of these and stack them together
inside a small beaker. Weigh the whole accurately on the fine
balance ; transfer the bulk of the contents of one of these tubes
to a hard glass flask (Kjeldahl flask), and replace the tube
carefully beside the others, weigh again, the difference represents
the .amount of meal taken for nitrogen estimation. Prepare two
such flasks.
MO Practical Physiology.
(2) The incineration. — Take, in a dry cylinder measure, 10 c.c,
of concentrated sulphuric acid and add it to the contents of the
Kjeldahl flask. Before pouring it in, allow a small quantity
of distilled water to run down the neck and inside walls of the
flask, and then while pouring in the acid rotate the flask gently
so as to spread the acid over the inside of the flask in a thin
layer ; this prevents the carbonaceous matter from sticking to
the wall of the flask. Add also 10 grm. or thereby of potassium
sulphate, and a small crystal of copper sulphate. Now place
the flask on a tripod in an inclined position in the fume
chamber and apply the flame, small at first and stronger later
on when all bubbling has ceased. After some hours, or less,
according to the ease of oxidation, the residue becomes clear.
Continue the heating for some time after it has become clear,
then allow the flask to cool on the tripod.
(3) Distillation of the Ammonia. — As already mentioned,
the H2SO4 in the Kjeldahl method oxidises the elements present
in organic substances and converts the nitrogen into ammonium
sulphate. Our object now is to break up this compound with a
fixed alkali, and measure the amount of ammonia evolved. When
the flask is quite cold, add about a hundred c.c. distilled water
(or less if the flask is small), and dissolve the crystalline residue
which has formed in the flask. It may be necessary to warm
the flask ; if so, it may be cooled again, under the tap this time,
when the residue is completely dissolved. Then prepare the
condenser, see that the water can run freely through its jacket
and that the tripod, bunsen burner, etc., are ready. Measure
out very accurately from a burette a sufficient amount of tenth
— or fifth normal acid (about 30 c.c. 1^ is enough for the amount
of meal taken in this experiment). Run this amount diractlv
into a very clean titration flask, add a drop of rosolic acid to
act as indicator in case the amount of ammonia which comes
over should be more than the acid taken can neutralise, and add
some distilled water till the flask is about a quarter filled.
Measure out roughly in a cylinder 100 c.c. of very strong
soda (23% or thereby), and have ready a long stemmed funnel.
Now add some water to the incineration flask so as to fill it about
one third full ; add also a spoonful of talc to prevent bumping ;
Practical Physiology. 141
then place the funnel in the mouth of the flask — incline it to one
side and pour in the 100 c.c. of strong soda so that it form a
layer beneath the acid contents of the flask. Then without
shaking more than you can help connect the flask to the con-
denser and apply the flame. The receiving end of the condenser
should be fitted with a short length of tubing which at the
beginning of the distillation should dip beneath the surface of
the tenth-normal acid in the titration flask. Later on when the
distillate comes over as condensed steam this may be taken out
of the acid fluid but the opening of the tube should never be far
from the acid, otherwise some ammonia may be lost. If the
amount of -^ acid taken at first has been insufficient, the dis-
tillate will become alkaline as shown by the pink colour due to
the indicator ; in such a case the estimation will still be good if
an exactly measured amount of the yg acid is promptly added,
sufficient to acidify the contents. The 100 c.c. of 23% soda is
usually sufficient when 10 c.c, H2SO4 was taken to begin with,
but of course more soda must be added if more HgSO,^ was added
to complete incineration. If the amount of soda added is suffi-
cient, the contents of the distillation flask become deep blue
when CUSO4 is present.
Once the distillation ^has begun, the distilling flask must not
be allowed to cool and must be kept connected to the condenser.
After three-quarters to one hour's distilling, test with litmus
a drop of the distillate as it leaves the tube — to see if ammonia
is still coming over. When the distillate no longer gives the
alkaline reaction, titrate the contents of the flask with -ro or |-
alkali. The difference between the amount of acid taken and
the amoimt of alkali needed to reach the neutral point in the
distillate is the datum for calculating the amount of ammonia
which has been distilled ofi. Suppose this difference is equivalent
to 20 c.c. of xo a^cid ; obviously the amount of ammonia which
this equals is 20 c.c. of ^ NH3 = 20 X 1.7 mlgm., or since it is
nitrogen and not ammonia we want, 20 X 1.4 mlgm in the amount
of substance taken, from which the percentage of nitrogen may
be calculated, and this X 6.25 = percentage of proteid.
The nitrogen in the excreta is estimated in a similar way
in samples of the whole urine and faeces separately in the dog.
or of the mixed excreta of the rat.
142 Practical Physiology.
(D) ESTIMATION OF FAT IN MEAL, &c.
First prepare a Soxhlet extraction apparatus fitted with
a condenser (internal form) and flaslc, on a sand bath which is
heated by a constant-level water bath.
Weigh an empty extraction thimble in a small beaker
accurately on the analytical balance, then fill it nearly full of
the meal and weigh again to obtain the quantity of meal taken.
Dry the meal and thimble as thus prepared at a temperature of
about 100° C. till next day. Place the loaded thimble in the
extraction apparatus and pour over it sufficient ether to syplion
over once and to come about half way up the syphon, then fit
the condenser in, and start the bath ; (keep the ether as far from
the burners as possible).
Allow the extraction to proceed for two days, then remove
the thimble and contents. Distill the bulk of the ether into the
space previously occupied by the thimble, but before it syphons
over into the flask disconnect it and syphon it over into the bottle
of "used ether." Have ready a clean, dry, weighed fat flask,
or other small glass vessel ; filter the contents of the fat flask
into this, washing out the flask and filter paper with several
small quantities of ether. Drive off the ether from the clear
filtrate on the water bath ; dry in a water oven, cool, and weigh.
The difference in the weight represents ethereal extract or "fat"
in the quantity of meal taken. Express the result in percentage.
The method of estimating fat in milk will be demonstrated
at this stage : — Ten c.c. of milk is allowed to soak into an Adam's
fat-free paper which is then dried and extracted with ether in
the same way.
(E) ESTIMATION OF CARBOHYDRATE.
Weigh accurately about two grammes of the meal, using a
small tube as described in the method of estimating the protein
(0,1). Put this into a flask capable of holding about five hun-
dred c.c. ; add about 200 c.c. of water and 7 c.c of concentrated
HCl (the percentage of HCl in the whole will then be about 1) ;
arrange the flask on a sand-bath heated by a bunsen and fit a
condenser in the mouth of the flask ; allow the flask to remain
Practical Physiology. 143
for one or two days on the hot sand-bath ; if the condenser is
efficient there will be no danger of the contents becoming
dry, but the apparatus should be examined from time to time to
prevent such a result. The ef!ect of this treatment is to hydrolyse
all starches present into glucose, which can now be estimated by
Fehling's method. Proceed as follows : — Cool the flask ; neu-
trahse the contents by shaking with calcium carbonate ; filter ;
wash the filter paper and any deposit which it may contain with
water several times, adding the washings to the original fluid ;
make the whole fluid up to 500 c.c. in a graduated flask ; mix
thoroughly and estimate the glucose by Fehling's method (276a)
without diluting futher. The results are only approximate
because reducing substances other than glucose are formed, and
the cellulose is not attacked. State your result in terms of starch,
e.g., if 87% of glucose is obtained from the meal, the original starch
would equal ^"^'"^^ = i|| X 87 = 78%.
The whole composition of the meal, etc., can now be con-
sidered ; add together the percentages of water, ash, protein,
fat and carbohydrate, the result should be 100, for the amount
of extractives is very small. In the case of oatmeal, the error
is more Ukely to be in the carbohydrate estimation than in any
other, owing to the cellulose.
(F) FEEDING OF ANIMAL, COLLECTION OF EXCRETA,
ITS PREPARATION FOR ANALYSIS, &c.
The animal (or animals) must be weighed daily at the same
hour, and then fed. Rats may be fed once a day, but larger
animals such as the dog, twice.
The amount of food necessary requires some knowledge of
its composition, and should be so arranged that the daily diet has
(1) a sufficient amount of protein ; (2) a sufficient caloric value.
For man this is accomplished by giving from 50 to 100 or more
grammes protein daily, and a total caloric value of 40 Calories
per kilogramme body weight, but for small animals the latter
factor must be greatly increased because of the relatively larger
surface in proportion to weight. The caloric value must also be
increased if the external temperature is low, and if muscular
Hi Practical Physiology.
work is being performed by the individual. Generally speaking,
about 3 or 4 grammes proteid daily, and 40 to 60 Calories is
sufficient for a medium sized rat of 200 grammes.
A dog thrives better on a fairly high protein intake, so it is
usual to allow them about 100 grammes protein per day, and 50
to 60 Calories per kilo.
The animal (or animals) must be kept at as equable a tem-
perature as possible during the experiment, and dogs should be
given a certain amount of exercise daily. The rats' cages should
be placed within a wooden box over the laboratory ovens, as
temperature affects them much more than large anin^als.
Having determined what the daily diet shall consist of, i.e.,
so much oatmeal and so much dried protein such as dried casein,
these amounts should be weighed out, mixed, and made up into
packets, one for each day of the experiment.
When required, one such daily ration is placed in the feeding
dish and about twice its weight of boiling water poured over it
to make a soft pulpy mass. When the amount necessary for
this has been determined, the same amount should be used each
day so that the meal is cooked each day to the same extent. Any
food left out over from one day should be separately analysed
or else added to the ffeces as if it had been eaten, but not absorbed.
The total Aveight of the excreta should be taken where rats are
used, or the volume of the urine and weight of the faeces when
a dog is the subject of the experiment. To obtain the whole
twenty-four hours' urine, dogs must be catheterised at a certain
hour when the experiment starts ; this urine is to be thrown away
but all the urine obtained during the experiment is to be kept
and the dog finally catheterised at the same hour when the
experiment ends. If a suitable collecting cage is available,
and if the animal is not too particular in its habits, these two
catheterisations may be sufficient, otherwise it should be
catheterised twice or three times daily. In rats this procedure
is impracticable, and the usual method adopted is to collect the
urine for four to six consecutive days, analysing every two days'
excreta together.
Treatment of the excreta. — In ike dog, the volume and
specific gravity of the urine may be noted and the whole mixed
Practical Physiology. 145
urine of one day made up to a definite quantity (500-1000 c.o.)
with water ; an aliquot portion of this is then taken for nitrogen,
phosphorus, etc. ; the size of this sample will depend on the
element being estimated, and the amount of it expected to be
present ; thus a dog receiving 100 grm. protein daily, if on
nitrogen equilibrium, excretes the nitrogen corresponding to that
amount, say 16 grm. ; suppose the urine to contain 15 grm. of
this, then if it is diluted to 1000 c.c, 5 c.c. would contain j^-fg grm.
^ .075 grm. nitrogen, and since 1 c.c. of ^e acid equals 1.4 mlgrm.
(.0014 grm.) nitrogen, one would require to use more than 53.6 c.c.
of tenth-normal acid, or 26.8 c.c. of -J acid ; such quantities are
easily worked with. The 5 c.c. sample should be measured
accurately in a pipette of the bulb form with only one mark,
viz., that at 5 c.c, and the last drop may be procured by blowing
through the pipette. It is inadvisable to wash out these pipettes ;
they are graduated to deliver 5.cc.
The fasces passed by a dog should be collected, mixed, and
spread in thin layer on a sheet of glass of known weight, dried
over a water bath or hot water pipes, weighed, and the dried
faeces scraped off, powdered if necessary (a coffee mill does this
very well), and sampled as described in the estimation of protein
(C. 1).
The excreta o/ a Rat consists of mixed urine and faeces ;
empty the whole into a flask capable of holding 500 c.c. or more ;
wash the shelf of the cage, the wire netting of the bottom, and
the collecting dish by directing a fine stream of distilled water
from a wash-bottle against these parts, aiding this by rubbing
any dirty places with a glass rod armed with india-rubber: When
everything has been transferred to the flask, add several c.c. of
sulphuric acid, mix and heat gently till all the pellets of faeces
become broken up ; this may be accelerated by vigorous shaking ;
when the fluid has acquired a uniform consistence, cool it, pour
it into a 500 c.c. graduated flask, wash out the flask used for the
mixing and add the washings to the main bulk, then add water
up to 500 c.c, mix, and empty the whole into a clean dry bottle.
This material can now be used for analysis, but before taking a
sample for nitrogen, etc., the contents of the bottle must be
thoroughly shaken and the flask or pipette filled before the
heavier particles have time to settle, Knowing the amount of
146 Practical Physiology.
protein given daily to the rat, calculate, in the way described
abovp for dogs, the amount of this fluid which should be taken
for nitrogen estimation so that the amount of -^ acid shall not
exceed 50 c.c. or that of |, 25 c.o.
(G) ESTIMATION OF PHOSPHORUS (as P,OJ IN THE
FOOD AND EXCRETA.
Place a measured amount of excreta or quantity of dried
substance weighed as directed in C.l in a Kjeldahl flask ; add
10 c.c. of a mixture of concentrated sulphuric and nitric acids,
equal parts ; and proceed to incinerate in the same way as in
the Kjeldahl process. When the flame is applied or even in the
cold, red fumes of NO2 develop so that the flask must be placed
in a fume-chamber with a good draught. Have ready a funnel
provided with a glass stop cock and filled with the acid mixture ;
this should be supported in a stand so that the end of the funnel
is over the mouth of the flask, the flask being inclined as in the
Kjeldahl method. When the red fumes cease to come off, add
some of the acid mixture drop by drop and continue heating and
adding drops of acid till the contents of the flask become and
remain clear ; allow the clear residue to heat for twenty minutes
to half an hour longer to ensure complete oxidation of the organic
matter ; then let the flask cool completely ; add a little water
and again heat to boiling to drive off the excess of nitrous acid.
So far the process has been merely that of incineration (by
Neumann's method) ; the older method of incineration was
similar to that described in (13) except that a very large excess
of sodium carbonate was added, the residue after incineration
was cautiously acidified. The next step in the process consists
in the precipitation of the phosphates by molybdate of ammonia.
Supposing we have done the incineration by Neumann's method,
make up the contents of the flask to 150 c.c. with water. This
may be done in the same flask or, better, in a beaker marked at
the 150 c.c. level ; if the latter is used, the flask must be rinsed
out several times into the beaker ; add 50 c.c. of 50% ammonium
nitrate solution, heat to 70° — 80° C, and then add 50 c.c. of a
10% ammonium molybdate solution ; the result is a yellow
precipitate of phospho-molybdate of ammonia which includes
Practical Physiology. 147
all the phosphorus in the fluid. Neumann has described an
accurate method of completing the estimation by titration, but
the older method of converting the phospho-molybdate into
triple phosphate (Mg(NH_j)P04) is more instructive for students'
work.
After standing for some time (15 minutes or longer) filter
ofi the precipitate, wash it several times with a weak nitric
acid till the washings no longer give any precipitate on testing
with ammonia. Then dissolve the yellow precipitate in ammonia
(1 in 3), and add 20-30 c.c. magnesia mixture. A precipitate of
ammonio-magnesium phosphate forms ; allow to stand for 12-
24: hours ; then filter through an ash-free filter paper : wash
chlorine-free with 1 in 3 ammonia ; dry and incinerate in a
platinum crucible. On incineration magnesium pyrophosphate
results (MgoPoO,) from which the amount of phosphorus
present in the original sample is estimated. The crucible is
weighed with the ash and its own v/eight subtracted. In using
a platinum crucible be careful not to allow the hot platinum
to touch anything but platinum, therefore use a platinum wire
triangle to support the crucible during incineration.
(H) THE CARBON BALANCE.
The carbon balance is more difficult to determine exactly
than that of nitrogen, etc.
It requires an air-tight chamber into which the animal's
cage can be put, some means of drawing air through this for
ventilation purposes, and means of sampling the current of air
and so estimating the amount of CO 2 produced. This gives the
great bulk of the carbon output on a carbohydrate and fat diet ;
the amount in the urine can be estimated from the nitrogen as
the carbon and nitrogen are present in fairly definite proportion
to each other (this ratio would be quite definite if urea were the
the only nitrogenous organic constituent in urine).
The amount of carbon in the food may be calculated from
the protein carbohydrate, and fat, or may be estimated by the
bomb calorimeter, where combustion is very complete ; the
contents of the bomb being allowed to escape through soda lime
and weighed.
148 Practical Physiology.
(Dem.) (Fifj. 16). The CO, for short periods, and small animals
mav be done by Haldane's method. The animal is placsd in a small
air-tight vessel which can be weighed with the animal inside.
Air is drawn through this by means of a filter pump, and the
current is made to enter (l)a soda-lime bottle to remove the COo
of the atmospheric air ; (2) a drying bottle of CaCU, or of sul-
phuric acid on pumice stone ; (3) the space in which the animal
is confined ; (4) a bottle with H,S04 dijtributed over pumice
stone ; (5) a soda-lime bottle, and (6) another H2SO4 bottle.
The H2SO4 bottle (4) absorbs the moisture given off by the
animal ; the CO2 is taken up by the soda lime, and the last
HoSO^. bottle absorbs any moisture derived from the soda lime.
Before the actual experiment begins, the following weights
are determined • — (1) the weight of the animal in its air-tight cage,
the connecting tubes (AA') being clipped at a given time; (2)
the weight of the HjSO^ bottle No. 4 ; (6) the combined weight
of the soda lime and HoSO^ bottles, Nos. 5 and 6. The bottles
and animal chamber are then securely connected and the current
of air drawn through for a fixed time reckoning from the time
when the tubes of the animal chamber were clipped before
weighing ; this time may be half to one or more hours according
to the capacity of the absorbing bottles. At the end of this period
the three weighings mentioned above are repeatd, and so one
obtains the CO 2 and water output in grammes for a given time ;
the temperature of ihe animal chamber and the weight of the
animal should also be taken, so that the results can be expressed
per kilogram, body weight.
Another important datum can be obtained from the weights,
viz., the "oxygen absorbed;" this is given by the difierence
between the loss of weight of the animal during the experiment
and the combined gain of the absorbing bottles (due to COj and
HoO). Some of the oxygen of the inspired air goes to oxidise
elements other than carbon, such as sulphur, phosphorus,
hydrogen, etc., and this oxygen is retained in the body of the
animal, and hence its loss of weight and the gain in weight of
the absorption bottles do not coincide.
If time permits, the experiment should be repeated at a
different temperature, or under other conditions capable of
affecting the CO 2 output.
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150 Peactical Physiology.
(I). THE LIME AND MAGNESIA BALANCE,
The Calcium intake and output may be obtained by esti-
mating the amount present in food and excreta as follows :-^
Incinerate a weighed amount, say 5 grm. meal in a porcelain
crucible after adding some dry sodium carbonate ; the inciner-
ation may be hastened at the end by adding small pinches of
potass, nitrate (as in 13) ; allow it cool ; dissolve the ash com-
pletely in dilute HCl, and filter. Add sufficient ammonia
to make slightly alkaline, and then acetic acid till the fluid is
distinctly acid. If a flocculent precipitate of phosphate of iron
remains undissolved in the acid fluid it should be filtered off ;
then add ammonium oxalate till no more precipitate appears,
heat to near boiling and allow to stand 24 hours. Then filter
through an ash-free filter paper, wash with water till the
washings are chlorine- and phosphate-free, dry, incinerate, and
weigh ; the result is CaO in the amount taken. Treat samples
of the excreta similarly. In dealing with urine it is best to
evaporate it down in a fairly large porcelain crucible and in-
cinerate in the same way.
The Magnesium in food and excreta can be estimated in
the filtrate from the calcium oxalate as above obtained. Con-
centrate the filtrate and washings,; filter if necessary, add some
sodium phosphate and excess of ammonia. The result is a
precipitate of triple phosphate which is treated in the same wav
as described under phosphate estimation, i.e., the fluid is filtered
after standing for twenty-four hours, the precipitate washed
chlorine-free with 1 in 3 ammonia, dried and incinerated in a
platinum crucible. The amount of magnesium is calculated
from the weight of magnesium pyrophosphate (Mg.jP20,)
which results.
(J)-
The Chlorine balance may also be estimated by incinerating
the food and excreta with a large excess of sodium carbonate,
dissolve the residue in dilute nitric acid and estimate the chlorine
in the solution by Volhard's method (267).
Practical Physiology. 151
INTERNAL SECRETIONS.
(306) Examine slides illustrative of the structure of the
following glands : — thymus, spleen, suprarenal, thyroid, pituitary,
pancreas, and sexual organs.
(307) Pith a frog ; excise both eyeballs carefully ; place
one in each of two watch glasses filled with normal saline ; note
whether there is any difference in the size of the two pupils ; add
to one a few drops of dilute adrenalin chloride ; continue to
observe the size of the pupils ; the one in the adrenalin will
become larger (dilate) ; (stimulation of the sympathetic nerve
supply to the iris causes a similar result, compare to exp. (195)
where the sympathetic was paralysed by section).
(308) Take a muscle curve in the usual way from a
gastrocnemius, sartoriiis, or hyoglossus, then inject adrenalin
into the muscle and repeat the curve ; increase in the height and
duration of the contraction usually occurs.
(309) Addition of adrenalin to the saline perfused through
the blood vessels of a frog causes a diminished flow due to vaso-
constriction. Since the frog is pithed this must be a peripheral
effect. Do this experiment as described in (196).
(310) Examine some dried thyroid substance for the presence
of iodine as follows : — Add a little water to the dried substance
in a porcelain crucible ; then add about twice the bulk of the
substance of pure solid NaOH ; apply heat, gently at first to
drive off the water, then more strongly till the whole mass is
charred, then add cautiously a few particles of potassium nitrate
to accelerate the oxidation, and continue heating and adding
the nitrate till the mass just becomes white ; avoid the use of
more potassium nitrate than is necessary. Cool the residue ;
dissolve it in a little hot water ; filter and cool the filtrate ; then
acidify with H2SO4, keeping down the temperature by holding
the tube under the running tap ; add a few c.c. of chloroform
and shake ; if iodine is present in the substance originally taken,
it is converted into an iodide by the oxidation in the presence
of an alkali, and is then set free by the acid ; the chloroform
dissolves out the iodine and assumes a pink colour when the
result is positive. Excess of potassium nitrate during the
incineration may lead to formation of iodates, and so vitiate
the result.
CHAPTER XI.
THE
NERVOUS SYSTEM AND SPECIAL SENSES.
CENTRAL NERVOUS SYSTEM.
(311) Examine the slides illustrative of the general structure
of the spinal cord and brain.
(312) Reflex action in the Frog. — Pith the brain of a frog
in front of a line drawn between the anterior margins of the
tympanic membranes, so as to leave the optic lobes and spinal
cord intact ; suspend the animal from the myograph stand by
means of a pin passed through the snout so that the lower limbs
hang free, and make the following observations : — (a) Note
that pinching the toes on one side causes reflex withdrawal of
that foot ; (b) an electric shock or other form of stimulation,
e.g., thermal, has the same effect ; if the stimulus is strong enough
the excitement spreads to the other foot, fore limbs and whole
body; (c) a "purposive" reflex movement may be elicited by
applying to the skin of the abdomen a small piece of filter
paper soaked in very dilute acid — the limbs are moved in such
a way as to remove the irritant ; wash away the acid from the
skin of the frog ; and {d) find the reflex time by Turck's method,
that is, allow one foot to dip into a beaker of very weak acid
and count the number of seconds that elapse before it is with-
drawn ; repeat with a stronger acid after washing ; the time varies
with the strength of the stimulus, but only within certain limits,
(e) Inliibition of reflexes. — Cut the head of the frog across so
as to expose the optic lobes or medulla ; take the reflex time by
Turck's method for a certain strength of acid ; place a crystal
Practical Physiology. 153
of common salt on the cut end of the medulla, or optic lobes ;
after a minute or two find the reflex time again with the same
acid ; it will be found to be much longer and sometimes the reflex
is abolished ; (/) Another method of inhibiting reflex action
may be tried on another frog — take the reflex time, tie a string
tightly round one fore limb in order to cause a strong afferent
impression, and again test the reflex.
(313) Prepare a frog as in (312), inject a few minims of a
strychnine solution^; and note that convulsive movements can
now be set up by very slight stimulation of the skin, or even by
tapping the table ; ■ pith the cord — the reflexes and convulsions
will disappear.
(314) Test your own knee-jerk by crossing one leg over the
other so that the former swings freely from the knee downwards ;
tap the patellar tendon smartly and note the involuntary forward
movement of the leg and foot.
(315) Simple Reaction Time (dem.). — This may be measured
with the pendulum myograph ; arrange the knock-over key as
part of the primary circuit of an induction coil ; the electrodes
of the secondary circuit may be placed on the skin (reaction
time for pain), or connected with a telephone (for hearing), or
the knock-over key may be made part of a galvanic circuit which
operates an electro-magnet and pulls down a lever with a white
disc attached (reaction time for sight) ; the point of time when
the signal is given is shown on the moving surface by a dip of
the writing lever due to the electro-magnet C (Fig. 17) being
thrown into action ; the lever remains down till the response
is given by opening key B. Take the time value of the moving
surface with the tuning fork as usual.
SPECIAL SENSES.
(316) Examine slides illustrative of the structure of the special
sense organs — eye, ear, olfactory mucosa, taste buds, nerve
terminations in skin, muscles, etc. If opportunity offers dissect
the eye of an ox, or other animal according to the directions
given in Stewart's Physiology, or in manuals of Practical
Anatomy.
1541i^'^P X gPRACTiCAL Physiology.
3
-O
Fig. 17.
Arrangement for measuring reaction time for sight. A,
knock-over key opened by the swing of the pendulum myograph ;
B, response key. These should be in separate rooms, or as far
apart as is practicable.
(317) Test the visual acuity of each of your own eyes with
Snellen's Test Types at 30 and 20 feet. Study the relationship
of the size of the characteristic parts of the letters to the distance
at which the letter is recognised by a normal eye, and calculate
the visual angle.
Practical Physiology. 156
(318) Observe, in a companion's eye, the Purkinje-
Sanson images seen as reflections from the anterior surface of
the cornea, anterior surface and posterior surface of the lens. Aslc
the subject to focus for a near object and then for a far one and
note whether you can see any change in the size and position of
the images. These observations can best be made with the
phakoscope of von Helmholtz, the use of which will be demon-
strated.
(319) Observe tjie reflex contraction of the pupil on stimu-
lation by a bright light, and its dilation in a darkened room or on
bhndfolding ; if one eye is blindfolded the other pupil dilates.
(320) (Dem.) Kiihne's Artificial eye illustrates many
important points in connection with the optics of the normal
(emmetropic) eye, and abnormal conditions of refraction. It
consists of a wooden trough which may be filled with water
tinged with eosin to show up the path of the rays. At one end
of the box is a curved glass window to represent the cornea ;
in some forms of the instrument this is covered with a water-
tight cap and plain glass face so that when the cap is filled the
cornea is thrown out of action ; the retina is represented by a
movable ground glass screen, and the lens and iris are also
represented by movable parts ; a ray of sunlight or other strong
light is sent horizontally through the cornea and the following
observations may be made, {a) The beam of light (parallel rays)
is brought to a focus at one point behind the cornea and lens
(= principal posterior focus) ; instead of using the whole beam of
light a piece of thin metal with a stencil cut in it may be inter-
posed, in such a case note that the image is reversed. Find the
position of the screen which corresponds to the principal posterior
focus.
(b) Remove the lens (cf. operation for cataract) and note
that now the rays are no longer focussed on the retina ; place
the removed lens in front of the cornea, it overcompensates the
defect and brings the rays to a focus in front of the retina, this
is because it is now acting with air on each side of it instead of
watery fluid ; find a lens that will compensate the defect and bring
parallel rays to a focus on the retina and then compare its strength
(focal length) with that of the lens which was removed.
156 Practical Physiology.
(c) Eeturn the lens to its place and abolisli the refractive
power of the cornea by filling the front cap with water, and pro-
ceed similarly to find a lens which will compensate for the defect.
(d) Remove the iris — the image may be seen to be less
distinct.
(e) Place the retinal screen further away from the cornea,
after replacing all the working parts, a blurred image results
comparable to myopia or short sightedness which is generally
due to elongation of the eyeball in the antero-posterior direction.
Correct the defect by a concave lens in front of the cornea.
(f) Proceed similarly to imitate Jtypermetrofia (long-sighted-
ness) by placing the retinal screen nearer to the cornea than normal
— correct by using a convex lens.
(g) Place a cylindrical lens in front of the cornea and note
the irregularity of the image due to increased refraction in one line.
This simulates astigmatism.
(321) Scheiner's Experiment. — Make two pin holes in a
piece of note paper at a less distance apart than the diameter of
your pupil ; stand with your back to the window and, holding
the perforated paper close to one eye, look through the holes at
a pin or needle held a foot or more away. At first you see a single
image of the needle ; then gradually bring the object nearer and
the image will be found to become double. If now the right hand
hole in the paper is blocked, the right hand image will disappear
and similarly for the left. Now place the needle about fifteen
inches from the eye and again look towards it through the two
holes, but accommodate the eye for distance as if you were looking
beyond the needle but yet paying attention to the image it pro-
duces : — A double image will again appear, and closing one of
the holes, say the right, causes the other image to disappear, in
this case the left hand one.
(322) Make three small pin holes arranged close together
in triangular form in a piece of note paper, hold a pin quite close
to one eye with the head up and hold the paper a short distance
(1-2 inches) in front of the eye ; at a certain adjustment of the
objects each of the three holes in the paper will show the head of
Practical Physiology. 157
the pin upside down. The pin in this case is held too close to
the eye to be focussed but yet it lies in the path of the rays
entering the pupil from the small holes ; each beam from a hole
casts a different shadow of the pin head on the retina in the erect
position but the mind misinterprets this and reverses the image.
(323) The Blind Spot : Mariotte's Experiment. Make a dot
and a cross about four inches apart on a piece of paper. Close
one eye, say the left, gaze steadily at the left hand mark, gradually
approximate the paper to the eye, at first both spots are seen but
at a certain distance from the eye the right hand one disappears,
to reappear again if the paper is brought still closer.
(324) The Yellow Spot. — Look at a bright white cloud through
a solution of chrome alum — a pink area will be seen — due to the
absorption of the blue green rays of the solution by the pigment
of the fovea centratis.
(325) The Ophthalmoscope. Indirect Method. — In an
otherwise darkened room, place a shaded light near to the
subject on the side of the eye to be examined ; the subject is to
be^^seated and the light placed opposite to or behind the ear so
that the face is in shadow ; seat yourself in front of the
subject close to and facing him, during the examination your
own eye should be about eighteen inches from the subject's eye ;
take the opthalmoscope in the right hand, place the back of the
larger mirror close to your eye and look through the central
aperture ; ask the subject to gaze steadily at a point in such a
position that the eye is rotated slightly inwards and then direct
a beam of reflected light into his eye through the pupil ; a red
appearance will be seen to occupy the pupil if everything is in
line ; when this is obtained, bring the convex lens into position
about 2Jin. in front of his eye, and in the path of the rays;
move the lens till the optic disc is found.
The direct method, which is more difficult for students' work,
will be explained.
Various other practical exercises on vision, and other special
senses, have been ommitted because the apparatus used can be
easily demonstrated, and the experiments are fully described in
the text books.
APPENDIX.
LiM of reagenli kept on the shelves of the working bench
required for one session are a\so given. From left to right I
the vpper shelf, the bottles are arranged as follows : —
Chemical Room.
Concentrated Pure Sulphuric Acid . .
Concentrated Pure Hydrochloric Acid
Fuming Nitric Acid
Acetic Acid, 10%
Glacial Acetic Acid
Oxalic Acid (crystals)
Alcohol, 96%
Alcoholic Potash . .
Amnion. Molybdate Solution
Ammon. Oxalate Solution . .
Ammou. Sulphate (crystals)
Ammon. Sulphide
Barfoed's Reagent
Barium Chloride, 10%
Calcium Chloride, 5%
Carbolic Acid, 5%
Chlorform
Copper Sulphate, 1 %
Ether ..
Fehling's Solution
Ferric Chloride, 2 %
Glyoxylie Acid . .
Guaiac Resin in Alcohol . .
Hydrogen Peroxide (watery)
Iodine 1% in 2% Potass, Iodide
Lead Acetate, neutral 10%
Lead Acetate, basic, sat. sol.
Litmus Solution . .
Magnesium Sulphate (crystals)
Magnesia Mixture
Mercuric Chloride, sat. watery soln. .
Millon's Reagent
alpha-Napthol in Methyl Alcohol
The amount
'.ginning irAth
u
oz.
1
oz.
li
oz.
3
oz.
H
oz.
1
oz.
3
oz.
3
oz.
3
oz.
3
oz.
4
oz.
3
oz.
3
oz.
3
oz.
3
oz.
3
oz.
3
oz.
3
oz.
3
oz.
1
oz.
1
oz.
3
oz.
3
oz.
3
oz.
1
oz.
3
oz.
3
oz.
3
oz.
3
oz.
1
oz.
Appendix.
159
1
oz.
3
oz.
3
oz.
3
oz.
3
oz.
3
oz.
3
oz.
2
oz.
1
oz.
3
oz.
1
oz.
3
oz.
10
oz.
10
oz.
10
oz.
10
oz.
10
oz.
10
oz.
10
oz.
Phenol-Phthalein in Alcohol
Picric Acid, sat. watery soln.
Potass. Feriicyanide, sat. watery soln.
Potass. Ferrocyanide, 5% . .
Silver Nitrate, 1%
Sodium Acetate -j- Acetic Acid
Sodium Carbonate, 10% . .
Sodium Chloride (crystals)
Sodium Hydrate (solid) . .
Sodium Phosphate, 10% . .
Sodium Nitroprusside, 5%
Sodium Sulphate (dry)
Uranium Nitrate, standard soln.
Hydrochloric Acid, 1 in 5 . .
Sulphuric Acid, 1 in 5
Nitric Acid, 1 in 5
Ammonia, 1 in 5 . .
Saturated watery Ammon. Sulphate
Esbach's Reagent
Potassium Hydrate, 10% . .
Sodium Hypobromite
Box of Litmus Papers ordinary and glazed.
[Where no quantity is mentioned the reagent is not to be put out or
made up till required. Unless where specially mentioned, the Acids to
be used are the diluted solutions in the larger bottles.]
Apparatus to be kept in the cupboard, or on the bench at each place : —
Iron Tripod with gauze top ; [the gauze to be heated to burn off
the dressing before the class meets.]
Pipe- clay Triangle.
Bunseu Burner connected by tubing to gas tap, [the burner should
have the gas exit narrowed or increased to give a flame two
inches long, and the air inlet should be capable of adjustment]
Porcelain Crucible (2J oz. capacity) and lid.
Crucible Tongs.
Porcelain Basin.
Mortar and Pestle.
Filter Funnel, filter paper (S. and S., 595, 11 cm.).
Filter Stand with clamp for burettes.
Two Burettes, with nozzle, tubing and chp.
Graduated Pipette (25 o.c. in ,l c.c.)
Measure CyUnder (100 c.c).
Thermometer.
Water- bath with wire netting cover.
Glass Rod.
Slides and coverglass.
Clean duster.
Test Tube Stand with test tubes | inch diameter and two large
test tubes (6x1 inch).
The microscopes are kept in the adjoining room.
160 Appendix.
Experimental Room.
Apparatus required for musc'.e work in general : —
Recording Drum.
Glazed Paper for drum.
Camphor on porcelain slab and handles for rotating the cylinder
in the flame, or luminous gas burner.
Myograph Stand with crank lever.
Frog, frog plate, dissecting instruments and dish for garbage.
Normal Saline (0.75% NaCl in tap water)
Thi'ead, pins, blotting yKiper.
Mounted Pin for pithing frog.
Duster.
Varnish (250 c.c. best white hard varnish -j- 1000 c.c. methy-
lated spirit -)- 10 c.c. castor oil).
Where Electric Stimulation is to be used : —
Daniell Cell with amalgamed zinc plate, copper plate, porous pot,
saturated solution of CuSO^, dilute (1 in 10) H0SO4.
Electric Wires, pin electrodes, emery cloth or sandpaper for clean-
ing ends of wires.
Mercury Key.
Induction Coil, Short-circuiting key.
Special apparatus^ material and reagctifs required for same of the experi'
ments. The material mentioned under one heading is frequently required
for those which fo'loif, and shou'd therefore he left on the bench till no longer
required^ e.g., egg white mentioned tender (15-20) is also required for tests
(25), (30), (32).
CHAPTER I.
(1-10). Minced tissue, e.g., moat, freed from fat as far as possible,
and dried on the water-bath ; ash-free filter papers (S. and S.
590, 11 cm.) ; mounted platinum wire for flame test ; satur-
rated watery solution of sodium hydrogen tartrate.
(11). Freshly made sat. watery ferrous sulphate.
(13) Dry sod. carbonate and potass, nitrate.
(15-20) Egg white pure and egg white diluted 1 in 5 with normal
saline and strained ; ^ — 1% gelatine ; 2% Witte's Peptone
in normal saline ; easeinogen solution made from milk or
solution of casein in 1% sod. carbonate ; globulin solution
(5% MgS04 extract of mince, strained); keratin shavings ;
(24) Tannic Acid 5% ; salicyl-sulphonic acid, saturated watery
solution ; Potassio-mercuric iodide solution ; trichlor-
acetic acid.
(26) Mixture of primary and secondary albumose.
(30) Parchment dialysis tube, 8 in. long ; tall jars ; blood serum.
(31) Live rat or guinea pig.
Appendix. 16 1
(32) Small test tube supported by a cork ring inside large test
tube ; water bath.
(33) 23% sodium hydrate ; undiluted egg white.
(35) Sodium nitroprusside.
(37-40) Water-bath.
Blood serum diluted 1 in 5 with normal saline.
(42) 2% Glucose.
(45) Bismuth subnitrate ; sod. carbonate, dry ; Nylander's
solution.
(47) Saffranin solution ; indigo- carmine solution.
(49) Phenyl-hydrazine, fluid ; 50% acetic acid saturated with
sodium acetate.
(50) Fresh brewer's yeast or active cake yeast ; fermentation
tubes.
(51) The spectro-polarimeter ; solution of glucose, 5 to 10%,
cleared if necessary by adding lead acetate, shaking and
filtering ; argand burner if sky is dull.
(52-55) Laevulose, pure, or inverted cane sugar; resorcin ; cane
sugar 2% ; maltose 2% ; lactose 2%.
(56-62) Starch powder, or raw potato, flour or other vegetable ;
starch mucilage 2% ; tannic acid 5% ; dialysis tube.
(63-66) Dextrin 2%.
(67-71) Glycogen solution of sufficient strength to be dis-
tinctly opalescent.
(72-73) Butter fat or suet ; Potass bisulphate crystals.
CHAPTER II.
(74) Thymus gland or pancreas, minced and soaked overnight
in ammoniacal water, and strained several times through
flannel ; artificial gastric juice (0.2% HCL + pepsin) ; ash-
free filter paper ; dry carbonate of soda.
(75) Cholesterin crystals ; chloroform ; or solution of pure
cholesterin in chloroform.
(78) Keratin shavings.
(80) Minced tendons of sheep's "trotters" extracted two or
more days with lime water and strained.
(81) Minced tendons; (82) Gelatine | to 1%; (83) Dried and
crushed bone ; 5% sulphurous acid.
CHAPTER III.
Frogs, frog plate etc., for exps. 85-113.
(85-92) Apparatus for muscle work as already described ; scale
pan and weights.
(93-98) Apparatus for electric stimulation with the monocord
instead of the induction coil ; thick wire for thermal stimu-
lation ; crystals of NaCl ; (99) The Rheonome ; saturated
solution of zinc sulphate; (103-104) Induction coil and
short-circuit key ; wires.
162 Appendix.
(105-108) Drums must be carefully overhauled at this stage,
driving cords adjusted, bearings oiled, stimulating pin and
brass strip for contact attended to, and see that the friction
pulley which transfers the motion to the base plate of the
cylinder is close to the axis ; tuning fork (simple or electrically
driven) ; test tube ; bunsen burner ; thermometer ; ice ;
extra stimulating pin for base plate.
(109) Fit up Mosso's ergograph, use the 5 kilo, weight and pass
the cord over the pulley in eeihng ; arrange a Sherrington
drum (slow speed) to take tracing. (110) Veratrin, saturated
solution in saline ; hypodermic syringe. (Ill) Usual appa-
ratus for muscle work and faradic stimulation with extra
stimulating pin. (112) Same with tetanus spring; seconds
clock working electro-magnet with writing lever, all on a
small tray for carrying round. (113) Glazed litmus paper
and usual apparatus.
(114-117) MgSOi extract of muscle (extract J lb. minced meat
overnight in 1000 c.o. of 5% MgSO^, strain several times) ;
general chemical apparatus including water-bath, thermo-
meter and test tubes for taking temperature of heat coagu-
lation of protein ; "extract of meat" on watch glasses.
(118-123) Solution of meat extract, 5%; ether; (124-127)
Ci-eatinin solution prepared from meat extract as given in
test (124) ; sodium nitroprusside, fresh, 5%.
CHAPTER IV.
(128-129) Frogs.; apparatus for muscle work and stimulation;
small cork chambers for applying COo to nerve ; putty or
soft sculptor's clay ; bottle containing marble chip and fitted
for the generation of CO 2 ; HCl ; switch commutator ; extra
pair of pin electrodes.
(130-134) Two extra Daniell cells; two simple keys; mono-
cord ; commutator ; non polarisable electrodes washed
thoroughly and soaked overnight in normal saline ; satu-
rated solution of zinc sulphate ; pipette ; arrangement for
supporting the non-polarisable electrodes. [This consists
of a glass rod, R in fig. 12, clamped in the split horizontal
limb of a small tubular T-piece, the vertical limb of which fit
into the open upper end of the vertical limb of the large
T-piece which carries the cork platform of the myograph ;
the electrodes are affixed to the glass rod by two bent strips
of spring brass perforated by holes for the rod about 1^ inches
apart, see fig. 12].
(135) Special electrodes covered with sponge or wash-leather;
5% salt solution, warmed.
(136-137) Usual apparatus for muscle work. (138) Pendulum
myograph and stimulation apjiaratus. (139-142) Tripods
with copper hook attached ; glass rod bent into shape of a
hook ; capillary electrometer and galvanometer.
Appendix. 163
CHAPTER V.
(143-146) Haemocytometers (Gowers' and Thoma-Leitz) ;
microsco)ic ; 3% sodium chloride solution ; 1% acetic acid
tinged with gentian violet ; paper for making calculation on ;
glazed litmus paper.
(147) Benzol and chloroform mixed to give a specific gravity
about 1060 ; choloform bottle ; benzol bottle ; large test
tube wide enough to admit hydrometer ; hydrometer ;
pipette to deliver drops of blood.
(148-149) Chloroform ; ether ; solution of bile salts ; 3 — 5%
NaCl.
(150-152) Jars containing blood which has been mixed when
shed with the following :— 27% MgSO.!, 1 part blood,
4 parts ; 0.4% Potass, oxalate in normal saline, 1 part
blood, 4 parts ; 3% sod. fluoride, 1 part^ blood, 9 parts ;
these mixtures should be let stand 24 hours to obtain some
plasma or the centrifuge may be employed for that purpose ;
blood serum ; water-bath, thermometer and usual chemical
reagents ; watery extract of minced liver or other tissue freed
from blood as far as possible. (153-160) Blood serum ;
dialysis apparatus ; Esbach's albuminimeter and reagent ;
watery HoOj or ethereal extract of same (= ozonic ether) :
ammon. sulphocyanide solution ; dried blood.
(161-168) Defibrlnated blood ; spectroscopes; luminous burners
fitted for giving the sodium flame (cut some narrow strips
of asbestos millboard, soak one end in strong NaCl solution,
dry thoroughly, attach to the burner by a flexible lead wire
so that the end of the asbestos strip just touches the flame) ;
Stokes' solution (2% ferrous sulphate in 2% tartaric acid,
ammonia to be added just before use till fluid is faintly
alkaline). (169) Haldane's modification of Gowers' haomo-
globinometer ; distilled water ; tube from gas tap connected
to fine glass tube for delivery of CO ; Haldane's apparatus
for estimating blood gases and oxygen capacity.
CHAPTER VI.
(170-171) Sheep's or ox's heart to each couple of students.
(172) Frog, frog plate, etc ; drum and apparatus for muscle
work ; heart-hook ; ice ; test tube and bunsen burner ;
seconds clock with writing lever ; myograph fitted with
straight lever and frictionless point, [the lever consists of a
fine straw about seven inches long ; about two inches from
one end a piece of aluminium foil is wrapped closely round
the straw and then transfixed by a piece of fine needle sharp-
ened at each end, this forms the axis of the lever ; the point
is made as follows : — cut a small triangular piece of alu-
minium foil, J inch wide at the base and 1^ inches long ;
roll the base of this triangle two or three times tightly round
164: Appendix.
a needle or pin which is just a trifle thicker than the pin
just to be mentioned ; replace the needle by an ordinary
pin 1^ in. long; in this way a kind of "pennon" is
formed with the pin as the staff round which the aluminium
can revolve easily as on a hinge ; coat the point of the pin
with colophonium or other cement and push it while still
warm into the open end of the straw lever till there is just
sufficient of it left for the hinge to swing on ; bend the alu-
minium into the arc of a circle ; the point {apex of the original
triangle) will then press against the surface of the drum paper
by the action of gravity and can be made to press more or
less heavily according as it is bent more or less acutely].
For experiments on heart work the shafting must revolve at a slow
rate : let the eyhuder revolve once in two minutes.
(173-175) Stethoscopes; cardio-graphs with recording tambours ;
sphygmographs (Dudgeon's and Marey's) ; papers for
sphygmograph ; camphor.
(176) Rabbit (weighed) ; paraldehyde ; small burette for
measuring dose ; cylinder measure, 100 c.c. ; distilled water :
perforated mouth gag ; stomach tube (flexible catheter and
funnel) ; animal holder ; dissecting instruments ; intra-
venous injection syringe ; manometer (mercurial or Hiirthle's
with connections to pressure bottle containing 2% sodium
citrate, and to a suitable Franck's canula ; ligatures ; adren-
alin.
(177) Capillary pressure apparatus.
(178) Martin's modification of the Biva-Rocoi apparatus for
estimating blood pressure in man connected by a side tube
to a recording tambour writing on a slow drum ; or Erlanger's
apparatus for the same purpose.
(179) Fit up an artificial scheme of the circulation with an enema,
syringe to represent the left ventricle and a long piece of
tubing to represent a vessel ; fix the bulb of the syringe between
two boards hinged together and acted on by an eccentric
worked from the shafting ; various instruments, sphygmo-
graph, cardiograph, manometers, stromuhr, etc , can be
demonstrated on the scheme, but for the stromuhr the
following arrangement is to be preferred : — Fix the jacket
part of a condenser in a vertical position, insert a cork with
a narrow tube in the wide lower end, leave the upper end open,
connect the tube in the lower end to the water tap, connect
the lower side tube of the jacket to the stromuhr and the
upper side tube to an indiarubber overflow tube, let water
run through the apparatus in amount sufficient to overflow
even when the stromuhr is being operated ; in this way the
rate of flow is made constant and can be regulated by a screw
el'p on the tubing leading to the stromulir.
(180) Mosso's Plethysmograph supported on a board swimg from
the easel or ceiling ; burette connected to interior of plethys-
mograph ; recording tambour and revolving drum ; see that
Appendix. 165
the indiarubber collar is in efficient working order and have a
large quantity of warm water (37° — 40°C) ready ; vaseline.
(181) Frogs; watch glasses. (182) Glass tube Sin. long drawn
to fine point.
(183) Gaskell's clamp arranged between two levers, the upper of
which is kept horizontal by a piece of very weak elastic ;
arrange both levers to write on a drum in the same vertical
line. (184) Sch fer's heart plethysmograph ; olive oil;
Locke's solution ; horizontal drum ; frog ; fresh defibrinated
blood may be required.
(185—192) Same apparatus as for (172); stimulation apparatus.
(193) Pilocarpine nitrate .5-10%, muscarine 5-10%, atropine
5-10%, nicotine 5%. all made with normal saline ; camel
hair brush for each drug. (19(i) Cylindrical tap funnel on
stand connected by tubing to fine canula to fit frog's aorta ;
adrenalin chloride (1 in 10,000) ; solution of amyl or other
nitrite.
CHAPTER VII.
(197-201) Stethographs, recording tambours or piston record ei' ;
stethoscopes ; spirometers of different kinds ; mercurial
manometer with scale on one limb, and mouth-piece connected
by tubing to other limb.
(202-203) Rabbit and apparatus as for (176), but instead of
manometer have a stethograph in the form of two cardiographs
(with the wooden shield removed) mounted on a bent strip of
lead so that the membranes of the tambours press on the two
sides of the thorax ; these are to be connected by a Y-tube to
the recording tambour.
(204) Haldane's Oas Analysis apparatus in working order ; tall
jar of water ; burette ; small cork for upper end of burette ;
bent S-shaped tube ; extra burette in stand, nozzle, and clip ;
solid NaOH ; duster.
(20.5) Leonard Hill's Blood Gas Pump ; Haldane's apparatus for
the same purpose ; defibrinated blood from the butcher, or
animal to be killed ; mercury ; pyrogallate of potash ; strong
KOH.
CHAPTER VIII;
(208) Have a large water bath ready, heated to 40° C. from the
night previous to experiment ; rabbit ; paraldehyde, etc., as
for (176) ; dissecting instruments ; drum, tambour, and small
baloon for recording movements. (209) Frog, frog plate, etc.;
myograph with straight lever as for (172) ; stimulation
apparatus.
(210-215) Starch mucilage 2%; porcelain slab; glass rod;
water bath, and usual chemical reagents,
166 Appendix.
(210-220) 0.2% HCl (7 c.o. concent. HCl in 1 litre water) ; Congo
red paper ; Gunzburg's reagent ; Boas' reagent ; jiorcelain
basin or broken piece of same ; burettes ; titration flask or
beaker, capacity about 300 c.c. (221-222) 1% lactic acid
solution ; ether.
(22:3-224) Pig's stomach, fresh; 0.2% HCl; raw meat or fish
minced, or fibrin, or partially coagulated egg white.
(225) Gastric digest (12 hours digestion of some of above material
in 0.2% HCl p'us some active pepsin).
(226) Fresh milk ; rennet ; water bath, etc.
(227-228) Fresh pancreas minced, let lie one day and extract with
1% sodium carbonate or ammoniacal water as described in
(227) ; protein for digestion as above (meat, fish, or egg) ;
tryptic digest prepared in similar way to gastric but use 1%
sod. carb. and active trypsin ; Bromine water (later). (229)
starch mucilage 2%. (230) Cream or neutral ohve oil ; fresh
pancreas ; (231-240) Ox bile ; cane sugar, dry ; sulphur,
in powder ; gall stones. (241-243) Gastric digest or 2%
Witte's Peptone ; starch mucilage ; olive oil ; 1% sodium
carbonate. (244-247) Intestinal mucous membrane dissected
off, minced, extracted 12 hours with water, and strained ;
cane sugar 2% ; lactose 2% ; maltose 2% ; yeast ; weak
solution of Witte's Peptone.
CH.^PTER IX.
Fresh normal human urine for tests (24S-271) arrange for supply of
pathological urines (248-2.54) Urinometers ; urea crystals, dry ; (272-280)
fresh sodium hypobromite (dissolve 100 grms. NaOH in 2.50 c.c. water and
add cautiously 25 c.c. bromine).
(255) Apparatus for quantalive es imation of urea in urine,
riz., small wide- mouthed bottle with paraffined cork or rubber
stopper perforated by a tube which is connected by rubber
tubing to an inverted burette in ;■■ tall jar of water ; small,
short test tube to fit inside bottle and marked at 5 c.c. level ;
jar to hold water for cooling the bottle ; cylinder measure.
(256-262) I'ric acid crystals ; urine with vM'ato deposit ; broken
porcelain for murexide test. (263-265) Fresh sodium nitro-
prusside ; hippurie acid ; herbivorous urine or human urine
containing indican ; 5% ' ' chloride of lime. ' '
(266) Apparatus for Nitngen Estimatlin, wi=., Kjeldahl flask ;
tripod, gauze, etc., in fume cupboard ; HjSOj ; KjSO^ ;
CuSO.j ; condenser ; titration flask ; fifth- or tenth-normal
acid and alkali ; two burettes ; 23°'o soda ; funnel ; talc ;
rosolic acid indicator.
(267) Apparatus for Chloride Estimation, viz., 100 c.c. and
50 c.c. flasks ; two burettes in stand ; standard silver nitrate
solution ; standard ammon. sulphocyanide solution ; ammonia
iron alum ; titration flask ; and the usual chemical apparatus.
Appendix. 167
(269) Apparatus for Estimation of Phosphates in Urine, rix..
Two burettes ; porcelain slab ; glass rod ; porcelain basin on
tripod with gauze ; standard uranium nitrate and sodium
acetate (Icept on the shelves).
(271) Bell jar to fit air tight on ground glass plate; (ripod to stand
inside bell jar ; two small porcelain basins ; milk of lime ;
tenth-normal alkali and acid in biircttcs ; vaseline.
(271a) Urines ^vith deposits, from hospital.
(272) Albuminous urine ; Esbach's reagent and albnminimeter.
(273) Urine containing blood ; spectroscopes ; burners to give
sodium flame, etc.. as for (161-1 (iS). (274) Bilious urine;
cane sugar ; flowers of sulphur ; Oliver's reagent (see text).
(275) Diabetic urine ; phenyl-hydrazine and sod. acetate in
acetic acid; yeast; polarimeter. (27'ia) Apparatus for
Estimation of Glucose (Fehling), viz., two burettes in stand ;
tripod, gauze, and porcelain basin ; 2.5 e.c. graduated (lipette
and cylinder measure for diluting the urine ; glass rod.
(27r)b) Apparatus for Pavy- Fehling Method, !'/z..the same as the
above butinstead of the porcelainbasin use a 300 e.c. flask with
a rubljer stopper, pierced by two holes, one for the nozzle of
the burette, the other for a bent glass tul)c to act as an outlet
for the steam ; put a screw clip on the tubing between the
nozzle and the end of the burette.
(277-279) Lactose solution '2";'„ ; phenyl-hydrazine, etc. ; |iure
pentose solution or solution of gum arable ; urine of rabbit or
dog after a dose of chloral. (280-281) Diabetic urine con-
taining aceto-acetic acid and acetone or an artificial solution
taining acetone.
(283) The cryoscope ; Beckmann's thermometer ; crushed ice ;
coarse salt ; urine.
CHAPTER X.
(284) Rabbit fed on carrots five hours before the class meets or
fresh oysters; scissors and force | is ; animal holder; large
porcelain dish with boiling water acidified witli! acetic acid;
large mortar and pestle : piece of wire gauze for removiiv.,'
pieces of lissup from the boiling fiuid ; clean sand; l;n-rrr
funnel and filler paper; tall jar; flruelci's rci^'ent; mctliy-
lated .spirit.
(285) Rabbit in collecting cage ; phloridzin.
(286-293) Fresh milk ; hydrometer ; ether ; rennet ; finely
grated cheese ; mortar and pestle.
(294-297) Hen's eggs ; spectroscope ; ether ; 10% NaCl ; 0.4%
HCl ; pepsin ; dried yolk ; ammon. sulphocyanide. (298-_
304) Flour ; muslin ; loaf of bread and other materials as
given in text.
(305) Two rats or one small dog ; suitable cages or kennel ; cath-
eter, if necessary, fitted up with wash bottle containinc
sterilised tap water ; several pounds of oatmeal ; or powdered
dog biscuit ; dried and powdered protein, e.g., casein.
168 Appendix.
A. Two pairs well-fitting watch glasses "with clips ; hot air
oven ; desiccator ; analytical balance.
B. Clean porcelain or platinum crucible with clay orTplatinum
triangle ; ash-free filter paper ; small beaker ; constant
level water bath.
C. Set of small, short test tubes, in light beaker, clean and dry ;
two Kjeldahl flasks ; strong H2SO4, etc., as for (266).
D. Soxhlet apparatus iitted with condenser and fat flask and
placed on sand bath over a. constant level water bath ;
extraction thimble ; Adams' paper ; fresh milk.
E. Flask to hold 500 c.c. or more fitted with condenser or long
glass tube to act as such ; sand bath ; apparatus for
glucose estimation (276a) ; CaCOj.
F. Ordinary balance for weighing up to several kilograms ; wash
bottle ; glass rod with indiarubber on end ; 1,000 c.c. or
500 c.c. measure flanks ; several bottles to hold one or
other of these amounts ; sheet of glass 12 x 10 in. for
drying dog's faeces.
G. Kjeldahl flask ; cylinder funnel with glass tap supported in
stand over flask ; mixture of concent. H2SO4 and HNO, ;
flask or beaker to hold 200-400 c.c. ; ammon. nitrate
50% ; ammon. molybdatc 10% ; platinum crucible and
triangle ; ash free filter paper ; desiccator.
H. Mouse ; apparatus for estimating GOj (Fig. 16) ; filter-pump
fitted to tap to draw air through apparatus ; place a gas-
meter between the pump and the apparatus.
1. and J. Platinum crucible and apparatus for Volhard's estima-
tion of chlorides (267).
(306-310) Frog ; watch glasses ; adrenalin ; apparatus for
general muscle work and stimulation ; hypodermic syringe in
working order ; thyroid gland substance ; potass, nitrate ;
chloroform.
(JHAPTEE. XI.
(312-315) Frog ; jars will) 1 in 300 aiul 1 in 1000 sulphuric acid;
acetic acid ; electric stinuilation apparatus ; seconds clock
fitted to bell ; sodium chloride crystals ; strychnine solution ;
hypodermic syringe ; narrow tape ; pendulum myograph
fitted for estimating reaction time (Fig. 17) ; telephone
receiver ; two electro-magnets with levers.
(316) Fresh ox eyes ; dissecting dish and instruments.
(317-324) iSncllen's Types ; phakoscope in dark room ; Kuhne's
artificial eye filled with water tinged with eosin ; mirror on
stand to reflect beam of sunlight ; set of concave and convex
lenses ; cylindrical lens to show astigmatism ; note paper and
needles ; solution of chrome alum in flat-sided bottles ;
ophthalmoscopes ; have tlie room darkened and argand
burners arranged for work with the ophthalmoscope.
Appendix. 169
Miscdlaneous Inslruciiois fgardiiifi the use of chemical apparatus.
BurnerS' — A bunsen burner gives most heat when it burns with a
non-luminous flame and without noise ; the hottest part ia
just above the inner mantle of the flame ; if it is desired to
diminish the flame, the air inlet should be diminished at the
same time.
Buiettes. — The correct way to read a burette is as follows : —
Remove it from its clamp and suspend it from near the top by-
holding it between the adjacent sides of the forefinger and
middle finger so that it swings freely and assumes a vertical
position ; fix some distant point of view on a level with the
eye, such as the roof of a house and bring the burette in front
of the eye so that the top of the column of fluid ia in line with
the point chosen, and then read the level at which the bottom
of the meniscus stands ; after running out the required amount
of fluid allow a minute or so to elapse in order to allow the
fluid which wets the inside to run down, and again read the
level in the same way. Always see that the nozzle of the
burette is full before you begin to run out the measured
amount.
Pipettes. — Since a pipette is filled by suction, it .should not be
used for volatile or corrosive liquids such as ammonia, strong
acids, or alkaline media such as Fehling's solution. When a
a sufScient amount of the fluid to be measured has been di-awn
up into the pipette, close the upper end with the tip of the
forefinger which ought to be quite dry ; read the level of the
fluid as described for a burette, and allow the requisite amount
to flow out by cautiously relaxing the pressure of the finger
on the upper opening of the pipette. Burettes, pipettes, and
measure flasks are used for exact work, measure cj-linders on
the other hand are less accurate and are used only for rough
measurements.
Filtering. — In preparing a filter paper and funnel sec that the paper
is of a suitable size ; wheji in position it should not project
above the to]i of the funnel ; fold the paper twice so as to
produce the quadrant of a circle, but in making the second
fold let one edge project a little beyond the other and then
open out the wider of the two possible hollows, thus forming
a cone the apex of which is an angle greater than a right angle ;
such a filter will cling to the funnel by its upper edge and leave
a space between the lower part and the side of the funnel,
through which the filtrate can run. The end of the funnel
should not dip into the filtrate.
Before beginning the filtration, wet the paper thoroughly with fluid
of the same nature as you are about to filter, except in some
cases where a dry funnel and filter paper must be used {e.rj.,
in 267).
Test Tubes, Flasks, Beakers, etc.— When boiling a watery fluid
in a test tube, keep shaking it gently, and, if only in the interest
of the bystanders, never attempt to boil a Ion? column of fluid
170
^PPENDIX.
in a te^t tube by applying the heat to the bottom of the tube.
If such a quantity must be boiled, use a large test tube or
porcelain basin, or boil the fluid in the upper part of the tube
only. Always proceed cautiously in neutralising strongly
acid or alkaline fluids in a test tube, else the contents may be
shot out by the heat evolved.
Bohemian glass beakers and flasks (flat bottomed) do not stand
boiling well, unless the heat is gradually applied, the contents
shaken occasionally, and the moisture which forms on the
outside removed. Never heat a flask en a naked flame when
it can be avoided ; use a tripod covered with gauze to support
the flask.
Jena glass beakers and flasks withstand heat -better but the same
precautions should be used. Flasks containing deposits and
semi-tluid contents should be heated only on the sand bath
or water bath. Measure flasks are on no account to be heated.
Thermometers. — Do not subject n thermometer to a temperature
which may lise above that for which the instrument is gradu-
ated, ('.(/., a hot air oven may reach a temperature of 120° C.
and a thermometer graduated to lOU" or 110° C. would be
burst at that temperature.
Mixing and Translerring Fluids, &c. — To ensure the uniform
mixing of fluids in a test tube, cylinder measure or flask, the
mouth should be closed with the finger or ball of the thumb
and the vessel inverted several times in succession. In the
case of beakers and porcelain basins, use a glass rod to stir
the fluids.
When the contents of one vessel are to be transferred completely
to another, use several small quantities of fluid to wash out
the first into the second instead of one large quantity.
Comparison of metrical with British measures.
1 gramme =
1 cubic centimeter (c. c. ) =..
0.065 gramme =
0.06 c.c. =..
28.35 grammes =^ . .
28.42 CO. r=
1000 grammes (1 Kilogram) =
1000 C.C. (llitre) =
4.546 litres =
1 metre == . .
2J centimeters approx. =
15.432 grains
16.95 minims
1 grain
1 minim
1 ounce (avoir) =r 437.5 grains
1 fluid ounce = 480 minims
2.2 lbs. (avoir.)
35.196 fluid ounces
1 gallon
39.37 inches
1 inch
To convert degrees Fahrenheit into degrees Centigrade or vii-e versa,
use this formula.
Degree F. =
9 X degree C
-32.
INDEX.
Pace.
Abnormal acids in stomach 101
Absolute contractile force 48
Acceleration of Heart 92
Aoebo-aoetic acid in urine 12(i
Acetone in urine 1 26
Achroodextrin 20, 99
Acid albumin 12, 102
Acid haematin 77
Acids, action of, on protein fi, 12
Acids of gastric juice 101
Acrolein reaction 22
Adrenalin, effect of, on punil 151
After-load ' 49
Albuminates 13
Albumins 13
Albuminuria 120
Albumoses 9, 14, 103
Alcohol, effect of, on protein 8
Aldehydes 14, 22
Alkali, effect of, on proteit! 11, 12
Alkali-Albumin 12
Alkali-haematin 77
Alkaline, phosphates in urine 118
AUoxuric bodies in urine 115
Amidulin 20, 09
Ammonia in urine 119
ABtunon. sulphate, action of on
jroteins ft
Amylopsin 105, 108
Anelectrotonus 57
Anode, stimulation at "break"
with 63
Apex beat 82
Artificial eye, Kiihne's 155
Artificial respiration 97
Ascending current 64
dn Page.
Ash, percentage of 138
Astigmatism 156
Atropine, effect of, on heart 92
Automatism of heart 88
Balance of carbon 147
of energy 136
Barfoerl's test U, 15, 18
Bases present in urine 118
Bile 106-108
effect of, on heart 92
liigment in urine 122
salts in urine 122
Bili-c3'anin,-rubin,-verdin 107
Biuret test 6,9,112
Blind spot of retina 157
Blood in urine 121
Blood pressure 84-85
in man 85-86
Blood vessels, innervation
and perfusion of 93
Boas' test for HCl 100
Boettger's test 15
Bone, chemistry of 26
Bread, chemistry of 134
Bronchial breathing 91
Burettes, method of reading 169
Burners, hints regarding 169
Calcium in saliva 99
in tissues 2
in urine 119
ions in coagulation of
milk 104
l72
Index.
Page.
Calcium ions in coagulation of
blood 72
metabolism of 150
oxalate in urine 119
phosphate in urine 119
Cane sugar 18
Capillary blood pressure 85
Caramel 16
Carbohydrates 14
in food stuffs 142
Carbohydrate radicle in protein 6
Carbon 4
Carbonates in tissue 2
Carbonic oxide haemoglobin 76
Cardiac impulse 82
inhibition 91, 92
muscle, contraction of 90
nerves 90-92
Cardio-graph 82-83
Carniferrin 55
Casein 132
Caseinogen 9, 131
Cathode, stimulation at ' ' make' '
with 63
Cervical sympathetic, effect of
section of 93
Cheese 131
Chemical stimulation 35
Chemistry of respiration 95
of muscle 53-55
Chlorides in tissue 2
in urine 116-117
Chlorine balance 150
Cholesterin 24, 107
Choletelin 107
Choline 24
Chromo-protein i:i
Closing tetanus 6.")
Coagulation of blood 71-72
of milk 104, 106
of protein by heat 7, 11
"Coffee grounds" in vomit 104
Collagen 26
Colour reactions of protein 5-6
■ Commutator 60
Compensatory pause 89
Composition of blood 73
of milk 1,30
of urine 111
Page.
Conduction of heart beat 88
Conductivity of nerve 56-63
Congo red as test for free acid 100
Conjugated protein 13
Connective tissues 25
Constituents of milk 131
Contraction of striped muscle 44
of non-striped muscle 98
of cardiac muscle 90
"Contraction without metals" 67
Contraction period 47
Co-ordination of heart beat 88
Creatine 55
Creatinine 55, 115
Crenation of corpuscles 71
Cryoscopy of fluids 127
Crystals of protein 1 1
Curara poisoning 44
Daniell ceU 35
Dentine 26
Deposits in urine 119
Derivatives of proteins 13
Descending current 64
Dextrins 20, 21, 90
Dialysis 10
Diastolic pressure 85
Direct stimulation 44
Disaccharides IS
Distillation of ammonia 140
Double Conduction 66
Drum papers 27
Drum, use of 44, 45
Dudgeon's sphygmograph 84
E
Earthy phosphates
117
Eggs
132
Elasticity of muscle
32
Elastin
26
Electro-physiology
67-68
Electrodes, non polarisable 60, 162
Electrotonus 57, 65
Emulsions 108
Enterokinase 104
Index.
173
Page.
Enumeration of corpuscles 69-70
Epithelial tissues 25
Erepsin 105, 109
Ergograph 50
Erythrodextrin 20, 99
Estimation of albumin in urinel20
of ash 138
of carbohydrate 142
of chlorides 117
of fat 142
of glucose 123, 124
of haemoglobin 78
of HCl in gastric juice 101
of phosphates in urine 118
of phosphorus in food
stuffs 146
of protein in food 139
of protein in serum 73
of urea 112, 113
of water percentage 138
Ethereal sulphates 115
Euglobulin ' 73
Excitability of nerve 56, 63
Excreta, treatment of 144
Extensibility of muscle 27-32
of contracted muscle 48
Extra systole 89
Extract of meat 54
Extractives of blood 74
Eye, Kiihne's artificial 155
Faradic stimulation 38-40
Fats 21
Pat of Cheese 132
of food-stuffs 142
of milk 131,142
of yolk 132
Fatigue of muscle 50
— ^ , of nerve 66
Fehling's test ' 14, 15
method of estimating
glucose 124
Fermentation test 16
Ferments, action of 13
Fibrinogen 73
■filtering, hints regarding 169
P
.lour
134
Page.
Food stuffs, chemistry of 130
Frictionless lever point 163
Frog's heart, beat of 81
Fruits 135
Full saturation 8
G
Galactose 18, 19
Rail stones 107
Galvani's experiments 67
Galvanic stimuli 35
stimulation of heart 58
stimulation of sensory
nerves 38
Gases of blood 96
Gaskell's clamp exp. 88
Gastric juice 100
Gastric digest 102, 103
Gastrocnemius-sciatic preparation
33
Gelatine
Globulins
Glycerine
Glycogen 21
Glycosuria
phloridzin
9, 26
9, 13
22
129
122
130
125
6
13
100
1.34
122
66
Glycuronic acid
Glyoxylic acid reaction
Gluco-protein
Glucose 20,
Gluten of flour
Gmelin's test 107,
Gracilis experiment
Graphic record of muscle twitch 44
Guaiac reaction 74, 96, 121, 130
Gvmzburg's teat for HCl 100
H
Haldane's method of estimating
haemoglobin 78
Halda'ne-Pembrey's method of
estimating CO^ 148
Haeraatin 77
Haematoporphyrin 77
Haemin 77
Haemochromogen 77
174
Index.
Page.
Haemoglobin 74, 75
crystals 11
estimation of 78
Half saturation 8
Hammerschlag's method 71
Heat coagulation temperature
of proteins 11
Heart, anntomy of 80
— effects of pole? on 58
influence of nerves 90-92
— ■ influence of drugs 92
vital properties of 87-90
Heart plethysraograph 89
sounds 82
Heavy metals, effect of, on
proteins 7
Heller's test for blood 122
for protein 7
Hetero-albumose 10,3
Hcxone bases 13
Hexoses 17
Hippuric acid 115
Histone 1.3
Hopkins' reaction 6
Huppert's test for bile pigment 122
Hydrochloric acid in stomach 100
Hydrogen 5
Hydrolysis of polysaccharides
19, 20
Hyoglossus preparation 50
Hypermetropia 156
Hypobromite test for urea 112
Indican in urine 1 15
Indiffusibility of protein 10
Indigo carmine 16
Indirect stimulation 44
Induction coil 38-39
Influence of galvanic stream
on conductivity of nerve 62
of load on muscle
twitch 47
— of poles on excitability
of nerve 58-62
of repeated stimuli 50
of varying temperature
on muscle curve 49
Page.
Influence of veratrin on muscle
curve 50
Inhibition of heart 91
— nf reflexes 152
Inorganic constituents of
tissue 1-4
Internal secretion 151
Intestinal movements 98
Intrapulmonary pressure 95
Inversion 18
Invertin 108
Iodine teat 19
in thyroid 151
Iodoform test for acetone 126
Iron in haemoglobin 74
in yolk of egg 133
Isomaltose 20
Jaffe'a test for creatinine 55
K
Kateleotrotonus
Keratin
Kjeldahl method
Knee jirk
57
25
116, 139
153
Lactalbumin and lactoglobulin
131
109
54
101
15. 18
Lactase
Lactic add (sarco)
in stomach
Lactose
— '- in urine 125
Laotosazone 18
Laevulose 17, 18
Laking of bloipd 71
Lasseigne's test 4
Latent period 46
Law of contraction 64
Lecithin 24
Legal's test for acetone 126
Leucin 105 120
Lever stop, use of 30
Lieberkuhn'a jelly U
Index.
175
Liver, glycogen in
Page,
129
Load, influence of, on muscular
contraction 47
Lutein of egg yolk 132
M
Magnesium in the tissues
balance
in urine
sulphate, action of,
protem
Maltase
Maltose 15, 18,
Marey's sphygmograph
Mariotte's experiment
Maximal stimuhia
Mechanical stimulation
Medium ciu-rent
Metabolism experiment 1
Metaprotein
Methaemoglobin
Milk
Millon's reaction
Minimal stimulus
Molisch's test
Monocord
Monosaccharides
Mosso'a plethysmograph
Mucin
of bile
of saliva
Murexide test
Muscarine, action on heart
Muscle, contraction of
, elasticity of
, extensibility of
Myograph stand
Myopia
Myosin
Myosinogen
N
Neef's hammer
Nerve impulse
Nerve-muscle preparation
2
150
119
1
10
109
20, 99
SI
157
40
SO
64
35-150
12, 13
76
130
5
40
6
36, 60
17, 18
87
25
106
99
114
92
44
32
27-32
34
156
53
53
-42
56
33
Page.
Neumann'.s method of esti-
mating PjO, 146
Neutral salts, action of
on proteins 8
on polysaccharides 19, 21
Nicotine, action on heart 92
Nitrogen in urine 116
in organic substances 4
Non-polarisable electrodes 60, 162
Non-striped muscle 98
Nuclein 23-24
Nuolein bases 54
Nucleo-proteins 13, 23
Nylander's solution ] 5
O.itnicf'l 13,5
Oliver s test for bile 122
Ophthalmoscope 157
Organic constituents of tissue 4
Organic sulpha tiw 115
Osazone 16
Ovo-globulin 11
Oxydases 96
Oxygen 5
Oxygen absorbed 148
Oxygen capacity 79
Oxyhaemoglobin 75
Pancreatic juice 104-106
Paradoxical cont.-.iction 65
Paramyosinogen 53
Patholoffical urine I2O-I27
Pavy-TVhling's method 15, 124
Pentoses 17, l.'i, I?.';
Pepsin IO2
Peptone 14, IO3
Peptonuria I27
Pe: fusion of blood vessels 93
Pettenkofe- 's test lOg
Piliicr'sjaw 64.
Phenylhydrazine test l^
Phlorid/.in ?lyccsuria 13o
Phosphates in tissue 2
in urine 117
176
Index.
Phosphates in metabolism
in meat extract
Phosphocarnic aci.l
Phospho-protein
Pilocarpine, action on ht"rt
Piotrowflii's test
Pipettes, method of using
Pithing of frogs
Plethysmograph
Point oi stimiilatio"
Polarimeter
Prlypep*'i.les
Poljsacchiri es
P-rtor's induction coil
Potassium in ash
ferrooynnide test
Page.
14li
51
55
13
6
IGO
28
87
46
17
14
19
39
2
8
9
103
.-1-21
Primary alb\imose
m gastric dii^cot
Prmciples. proximate
Prone pressure method of arti-
ficial respiration 97
Protamines 1 3
Proteins 5-14
Protein, aromatic radicle of 6
, carbohydrate radicle
of
colour tests for 5
precipitation tests for 7
action of ferments on 13
action of neutral salts
S
11
13
139
73
13
12
103
5-21
73
99
S3
94
115
155
152
119-120
, crj^stals
, derivatives of
, estimation of
of serum
. varieties of
Proteoses
Proto-alhumose
Pjoximate principles
Pseudo-globulin
Ptyahn
Pulse, the *
Pulse and respiration rates
Purin bodies in urine
Piu-kinje-Sanson im.ages
Purposive reflex
Pus in urine
Paoi.
Reaction of l^lood 71
of degeneration fiB
of muscle "i2
time 153
Reducing sugars 14
Reduction of haemoglobin 75
tests 14
Red corpuscles, estimation of fi9
Reflex time lo2
Reflex action 152
Refractory period 90
Relaxation of muscle 47
Rennin. action on milk 104. 131
Respiration 94-97
He.^piratorv movements 94
Respiratory sounds 94
Rheonome 37
"Rheoscopic preparation (i8
"Ring" method 7
Rnthei-a's test 1211
Salt'ranin 15
Salicyl-sulphonic acid S
Saliva 99
, action of, on starch 20
Salkowski's reaction 24
Saponification 21
Sarcolactic acic' 54
Sartorius prepni-ation 2S
Scheiner's exp. 15()
Schiff's test 114
Schlosing's method of esti-
matiu'^^NHj 119
Sclei'o-protein 13
Secondary albumose 9, 103, 127
Secondary contraction fiS
Separation of acids in stomacl'
contents 102
Servim globulin 9. 73
Short circuitintr key 44
Sino-auricular junction, stimu-
lation of 92
Snellen's types 154
Soap 22
Index.
177
Page.
Sodium in ash 2
Sodium silts, action of, on
protein, 10
Soluble starch 20
Sounds of heart 82
Sounds, respi'-'atory- 94
Soxhlct method of estimating
fat 142
Special sense; 15"
Specific gravity of blood 71
of urine 110
Spectroscopic examination of
blood pigment : 74, 121
Spectro-polarimetor 17
Speed of drum 44
Sphygmograms 83-84
Sphygmometer 85
Spirometer 95
Staircase effect 90
Stannius ligatures 89, 90
Starch 19
digestion 99
Steapsin ] 05
Stethograph 94
Stimulation of heart 89
of nerves in situ
, faradic
65, 66
38-40
32
75
Stimuli in general
Stokes' reagent
Stomach contents after test
meal 104
Stromuhr 86
Strong current 64
Submaximal stimuli 40
Subminimal .stimuli 40
Succus entericus 108-109
Sugar in urine 122
Sulphates in urine 115,117,118
Sulphooyanide in sahva 99
Sulphur in organic tissue 4
Sulphur in protein
Sulphur test for bile 107
Summation if stimuli 50
Superposition of contraction 51
Systolic pressure 85
T
Paqe,
Tannic acid 8, 19
, Temperature of heat coagu-
lation of protein 1 1
Temperature, influence of, on
muscle 49
Tetanus, genesis of 51-52
of heart muscle 90
Thermal stimulation 35
Thrombokinase 72
Thymus, nucleo-protein of 23
Thyroid, iodine in 151
Trommer's test 14
Tropaeolin test for HCl 109
Trypsin 104-105
Tryptophane 105
Tubular breathing 94
Tl'rck's method for estimating
reflex time 152
Tyrosin 105, 120
u
Uffelmann's test 54, 101
Unipolar induction 42
Uranium nitrate method of
estimating P..O5 118
Urates ' 113, 119
Urea 111-113
Uric acid 113-114,119
Vagi, ciTect of division of, ou
respiration 95
Vagus, effect on heart of 90
Varieties of carbohydrate 17
of leucocytes 70
of protein 13
Vegetable food stuffs 134
of nerve impulse 66
Velocity of blood 86
\'enous blood pressure 80
178
Index.
Page.
Veratrin, influeneo- of, o" mnsde
twitch
Vesicular breathing
VeR3f1s, innervation of
Visual acuity
Vital capacity
Vitellin
Vocal resonance
Volhard's method of estimating
NaCl
^ omited matter 104
Yi
Water percentage 138
Weak current 64
Weyl's test for creatinin 55
White corpuscles, enumeration
of 70
Work done by muscle 48, 49
50
94
93
154
Xanthin bases
Xanthoproteic test
114
5
95
133
Y
95
117
Yellow spot of eye
Yolk of egg
157
132
r_
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