A LABORATORY MANUAL 
OF 
Physiological Chemistry 
BY 
ELBERT W. ROCKWOOD, 8.5., M.D. 
Professor of Chemistry and Toxicology in the 
University of lowa 
With OfiE Cohcred Pehte hjid Three Piiates 
OF JVIICpOSCOPIC PpEPHpHTIOHS. 
PHILADELPHIA, NEWYORK, CHICAGO 
THE F. A. DAVIS COMPANY, PUBLISHERS 
1899 COPYRIGHT, 1899, 
THE F. A. DAVIS COMPANY. 
BY 
[Registered at Stationers’ Hall, London, Eng.] 
Philadelphia, Pa., U. S. A: 
The Medical Bulletin Printing-house, 
1914.-1(3 Cherry Street. PREFACE. 
In view of the results attained from the course given 
in physiological chemistry in this University, as well as 
the experience of others, the author is firmly convinced of 
the superiority of the laboratory method of instruction 
over the didactic, believing that it is only by practical work 
that the student can become familiar with the physio- 
logical changes in progress in the animal body and their 
products. This hook has been prepared with the aim of 
imparting accurate knowledge through the student’s own 
observation. It has seemed advisable to include with the 
directions for experimental work a brief explanation of 
the facts observed, so as to call attention to their meaning; 
or, at times, to state others which are important, hut which 
could not well he demonstrated in such a course as this. 
Some acquaintance with general chemistry and with 
chemical manipulation is presupposed. 
For the purpose of making the course flexible, the 
less important experiments, or those which are not of 
general interest, have been printed in smaller type. A 
few blank pages have been inserted for additional notes 
by the student. It has been found that the time usually 
assigned to chemistry in one year of a medical course is 
sufficient for the performance of most of the experi- 
mental work. 
As far as possible, the work has been so arranged as 
to require but a small stock of apparatus and reagents IV 
PREFACE. 
and such as arc readily obtainable. By this means a large 
class can carry on the work together. Complicated ex- 
periments have been omitted or put in small type for 
the use of advanced students or those who choose to 
spend more time upon the subject. 
The animal substances which are required—albumin, 
blood, bile, and others—can be found in the market or 
obtained from the slaughter-house. If no hospital is 
near, gastric juice, urine, etc., corresponding to patho- 
logical specimens can be prepared artificially for testing 
by the student. The expense of the course is very small. 
University of lowa, 
Elbert W. I’opr wood. 
July 31, 1899. TABLE OF CONTENTS. 
I INTRODUCTION 1 
PAGE 
The Carbohydrates 1 
Starch 4 
Dextrin 7 
Glycogen 8 
Cellulose 44 
Glucose 12 
Lactose 47 
Sucrose 49 
Maltose 49 
The Fats 20 
The Lecithins 20 
The Proteins . 28 
Albuminous Substances 30 
Albumins 35 
Globulins 33 
The Albuminates 39 
Albumoses and Peptones 41 
Fibrin 43 
Coagulated Albumin 43 
The Proteids 44 
Mucin 44 
The Nucleoalbumins 45 
The Nucleins 48 
Hsematogen 50 
The Albuminoids 50 
Collagen 50 
Gelatin •. . 54 
Blast in 53 
Keratin 53 
Fermentation 54 
The Saliva 58 CONTENTS. 
PAGE 
The Gastric Juice 02 
Gastric Digestion 04 
Methods of Testing Gastric Juice 71 
The Pancreatic Juice 82 
The Pancreatic Ferments 85 
Leuein and Tyrosin 87 
The Blood 89 
Composition 89 
The Corpuscles 89 
The Serum 92 
Clinical Tests 92 
The Coloring Matters 94 
Haemoglobin 94 
Oxyhaemoglobin 98 
Methaemoglobin 99 
Hsematin and Haemin 100 
Carbonic Oxid Haemoglobin 101 
Haemochromogen 102 
Hae m a t op or ph y rin 102 
Methods of Testing Stains 107 
The Bile 108 
Biliary Acids 112 
Cholesterin 114 
Biliary Pigments 115 
Bone 110 
Muscular Tissue 119 
The Plasma 121 
The Extractives 122 
Milk 124 
The. Urine 127 
Secretion and Physical Properties 127 
Reaction and Fermentation 129 
Urea 133 
Uric Acid and Urates 142 
Hippuric Acid 14G CONTENTS. 
Chlorids 147 
Phosphates 149 
Sulphates, Inorganic and Organic 152 
Albuminuria 156 
Globulinuria 159 
Albumosuria 159 
Peptonuria 159 
Fibrinuria 101 
Glycosuria 101 
Acetonuria 103 
Diaceturia 164 
Lactosuria 105 
Choluria 106 
Hsemoglobinuria and Haematuria 109 
Mueinuria 171 
Lipuria, or Chyluria : 172 
PAGE 
Urinary Sediments 173 
Pus 170 
Mucus 177 
Epithelium 177 
Blood 178 
Casts 178 
Bacteria 182 
Spermatozoa 182 
Uric Acid and Urates 183 
Calcium Oxalate 184 
Phosphates 185 
Calcium Sulphate 187 
Calcium Carbonate 187 
Tyrosin 188 
Eat 188 
Systematic Testing of Urine 188 
Urinary Calculi 191 
The Metric System 194 
Composition of Reagents 19G 
Index 199 PLATE I. 
1. a, Wheat-starch granules, 
ft, Potato-starch granules. 
2, a, Corn-starch granules. 
h, Buckwheat-starch granules. 
3. Haem in crystals, color brown. 
4. Cholesterin, coloi’less, transparent. 
5. Phenyl-glucosazone, yellow. 
0. a, Urea, colorless. 
I), Urea nitrate, colorless. PLATE I. PLATE 11. 
7. Calcium phosphate, crystallized and amorphous forms 
both colorless. 
8. Triple phosphate, “coffin-lid” crystals, colorless. 
Sodium urate, brown spherical masses with spicules. 
Bacteria. 
9. Ammonium urate, “thorn-apple” forms, color brown. 
Calcium carbonate, spherules and dumb-bell forms, 
colorless. 
10. Calcium oxalate, dumb-bell ’ and “envelope-shape” 
crystals, colorless. 
11. Uric acid crystals, yellow to dark brown. 
Amorphous urates, brownish. 
12. a, Calcium sulphate crystals, colorless. 
h, Impure leucin, nearly colorless. 
('. Tyrosin, colorless when pure. PLATE 11. PLATE 111. 
13. a, Normal pus-corpuscles or mucus-corpuscles, gran 
ular. 
It, Pus-corpuscles swollen with acetic acid, showing 
nuclei. 
c, Pus-corpuscles showing amoeboid movement. All 
colorless. 
14. Different forms of epithelial cells, colorless. 
15. Granular casts, colorless. 
d, Blood-corpuscles, nearly colorless. 
16. Epithelial casts, colorless. 
17. Hyaline casts. 
a, Broad or waxy, colorless. 
It, Narrow, colorless, and extremely transparent. 
18. a, Fat-casts. 
It, Yeast-fungi in urine, 
c, Spermatozoa. PLATE 111. PLATE IV. 
ABSOKPTION-SPECTEA. 
1. Oxyhsemoglobin. 
2. Haemoglobin. 
3. CO-hsemoglobin and CO-hsemochromogen. 
4. Methsemoglobin, alkaline. 
5. Hsematoporphyrin, acid. 
6. Hsematoporphyrin, alkaline. 
7. Hsemochromogen, alkaline. 
8. Hsematin, acid. 
9. Hsematin, alkaline. 
10. Sulphur methsemoglobin. 
11. Methsemoglobin, neutral or faintly acid. 
12. Pettenkofer’s test for biliary acids. PLATE IV INTRODUCTION. 
The principal materials which enter into the com- 
position of the animal body, as well as of the food neces- 
sary for its support, may he divided into several general 
classes:— 
1. Water. 
3. Mineral substances 
I. Inorganic 
1. Non-nitrogenons 
compounds, sneli as 
" (a) Carbohydrates, 
(ib) Fats. 
II Or anic 
3. Nitrogenous compounds, such, as the 
Proteins. 
There are a number of nitrogenous compounds in the 
animal body which cannot be classed under the proteins, 
and others which contain no nitrogen, but which do not 
belong to the carbohydrates or fats; nevertheless, these 
three classes include, by far, the largest part of the organic 
constituents. 
THE CARBOHYDRATES. 
The carbohydrates are composed of three elements: 
carbon, hydrogen, and oxygen. The latter two are always 
present in proportion to form water, and in this the carbo- 
hydrates differ from the fats, which contain less oxygen. 
The name of the group is derived from their composition, 
1 THE CARBOHYDRATES. 
although they cannot he made directly from carbon and 
water. Most of them contain in a molecule six atoms of 
carbon or some multiple of six. Many organic compounds 
of carbon, hydrogen, and oxygen, however, have the two 
latter in the proportion to form water, but do not belong to 
the carbohydrates. 
They are found in both the animal and vegetable 
kingdoms, but are most abundant in the latter. 
The different members of the group differ greatly in 
their properties, such as the power of crystallization, fer- 
mentation, reducing effect, action on polarized light, taste, 
etc. 
They may be divided according to their molecular 
composition into three classes:— 
I. Glucoses, or monosaccharids, COII12Oc, including: 
1. Glucose, or grape-sugar; also called dextrose. 
2-. Fructose, or fruit-sugar; also called lasvulose. 
3. Less important are galactose, mannose, and sev- 
eral others. 
11. Saccharoses, or disaccharids, CI„FI22011, includ- 
ing;— 
1. Sucrose, or cane-sugar. 
2. Lactose, or milk-sugar. 
3. Maltose, or malt-sugar, and some others. 
111. Amyloses, or polysaccharids, (C6HloOd)x, includ- 
ing:— 
1. Starch. 
2. Dextrin. 
3. Glycogen 
4. Cellulose; also a number of gums and others of 
less importance. THE CAEBOHYDEATES. 
3 
In addition to the above classes there are a number of 
compounds containing three, four, five, seven, eight, or 
nine atoms of carbon in a molecule. As yet they are not 
known to he of any great importance in physiological 
chemistry. 
The first class, the monosaccharids, is so named be- 
cause they contain one of the groups of six carbon atoms. 
They are mostly crystalline, easily soluble in water, and 
have a sweet taste. Chemically they are aldehyde or ketone 
compounds of the hexatomic alcohols. They have the 
power of reducing the oxygen compounds of the metals and 
of forming compounds with phenyl-hydrazin. They will 
also undergo fermentation with yeast. 
The disaccharids contain in a molecule twelve atoms of 
carbon. They may he conceived of as composed of two 
molecules of a monosaccharid minus one molecule of water: 
C 6H1206 -f- CgH12Og Cl 2H22011 -j~ h2o. 
By the action of acids or ferments they take up a 
molecule of water and form two molecules of a mono- 
saccharid. This formation of one or more simple sugars 
from a molecule of a disaccharid is called inversion, and 
the resulting sugar is known as invert-sugar. The di- 
saccharids do not undergo fermentation with yeast until 
they have been inverted. They are all soluble in water and 
have a sweet taste. 
The polysaccharids contain more than two groups of 
six carbon atoms, though the number is, in most cases, not 
positively known, and therefore is represented by x. They 
have probably a much higher molecular weight than the 
other classes. Their constitution is not known. They are 
mostly amorphous, insoluble in water, and, consequently, 4 
THE CARBOHYDRATES. 
tasteless. Ferments and acids convert them into the mono- 
saccharids. They do not have reducing power. 
Starch. 
Starch occurs in the cells of the plant. It is in the 
form of grains or granules, which vary in size in different 
plants from about 0.002 millimeter to ten times that. 
The granules are composed of two parts: an inner, soluble 
one, called granulose, and an outer one, called cellulose. 
This latter is insoluble in water and protects the starch 
from the action of many of the weaker ferments. When 
boiled or acted upon by alkalies it is broken, allowing the 
granulose to escape and forming starch-paste, or soluble 
starch. The shape and size of the granules differ so much 
in the different plants that the source can often he deter- 
mined by its microscopic appearance. Those of the potato 
have a shape somewhat similar to that of a clam-shell, those 
of wheat are round and smaller, and those of buckwheat 
more irregular. (Plate I, 1 and 2.) 
Starch can be obtained from the parts of the plant 
where it is stored up, like the tuber of tire potato or the 
kernel of grain, by macerating it, then washing out the 
starch with cold water. 
Starch is a colloid. A colloid is a substance which 
when dissolved will not pass through an animal membrane 
or parchment. They are the opposite of crystalloids, which 
are usually crystalline and which will diffuse through such 
membranes. This process of diffusion or separation of 
colloid from crystalloid substances is called dialysis. As 
starch cannot pass through an animal membrane, it must 
be changed to a diffusible form before it can be absorbed. 
This is effected by ferments in the saliva and pancreatic 
fluid. STARCH. 
5 
By heating to 160° to 200° starch is converted into 
dextrin.1 By boiling a solution with a dilute acid it is 
changed first into dextrin, then into glucose. Ptyalin 
changes it first into dextrin, finally to maltose. The dias- 
tase of malt gives the same products. 
Starch gives an intense-blue color with a solution of 
iodin. This color disappears on heating the liquid; but 
if it is not heated too long it becomes bine or purple again 
when it cools. The color will also be destroyed by the ad- 
dition of anything which will form a compound with the 
iodin, such as sodinm.thiosnlphate, silver salts, or the alka- 
line hydrates. 
Experiment 1.—Starch may be prepared from a potato by 
grating it upon a tin grater, stirring it up with a little water, 
and squeezing the water, Avhich contains a large part of the starch, 
through a piece of unbleached muslin. After repeating this with 
several portions collect the water in one vessel and allow the 
starch to settle to the bottom. Pour off the water, add more, and 
allow to settle again, repeating till the starch appears clean and 
white. Take what is needed for the experiments and let the rest 
dry. Enough starch for the microscopic examination can be 
obtained from the scrapings from a potato without washing. 
The cellulose-fibers will then be seen also. 
Experiment 2.—Examine the starcli under the micro- 
scope. Notice the shape of the grannies. 
Experiment 3. Place a drop of very dilute iodin 
solution upon the slide so that it runs under the cover- 
glass and notice the markings which are thus brought out 
upon the granules which are least colored. 
Experiment 4.—Examine in the same way starch 
1 Unless otherwise stated, all degrees of temperature will be 
understood as referring to the centigrade scale. THE CARBOHYDRATES. 
from other sources: corn, wheat, buckwheat, etc. Observe 
the difference in the size and shape of the granules. 
Experiment 5.-—Prove that starch does not dissolve 
in cold water by filtering after shaking powdered starch in 
a test-tube of water. lodin gives no color to the filtrate. 
Experiment G.—Add about a gramme of starch to 100 
cubic centimeters of cold water, mix it thoroughly, and 
hoik The starch has dissolved, as is shown by filtering and, 
after cooling, testing a portion of the filtrate with iodin 
solution. A deep-blue color is produced. It is destroyed 
by heating, hut reappears as purple, or blue again upon 
cooling. 
Experiment 7.—Tie a piece of animal or vegetable 
parchment over the end of a funnel, or use instead a piece 
of parchment dialyzing tube. Eirst see that this does not 
leak. It should hold water when suspended by the two 
ends. Place inside some of the starch solution made in the 
preceding experiment and hang the whole in a small beaker 
of water, so that the liquids inside and outside are at the 
same level. Allow it to stand several hours, then test the 
water outside with iodin for starch. It does not pass 
through because it is a colloid. 
Experiment 8.—Examine under the microscope the 
starch-paste which has been made by heating starch in 
water. The granules have been burst open and destroyed. 
Prove that starch can he decomposed by acids or fer- 
ments by means of the following:— 
Experiment 9.—ln about 100 cubic centimeters of 
water in a porcelain dish boil enough starch, previously 
moistened with cold water, to make a thin paste. Add 
about 10 cubic centimeters of dilute H2S04 and boil, stir- 
ring at first, until the liquid becomes thinner. Keep the 
solution up to its original volume by the addition of water. DEXTRIN. 
7 
If this is not done the strong acid will turn the liquid 
brown or black. From time to time remove a portion, cool, 
and test with iodin. When the iodin gives a red color the 
starch has been converted into dextrin. When no color 
appears on the addition of iodin it has been changed to 
glucose. Test a portion for glucose by adding an equal 
volume of sodium hydrate solution, then, drop by drop, 
cupric sulphate solution till a deep-blue color is produced. 
Heating this will give a yellow or red precipitate, showing 
the presence of glucose. This is known as Trommer’s test 
for glucose. 
Experiment 10.—Try Trommers test with the starch 
solution. It docs not respond. 
Experiment 11.—Add gradually to the remainder of 
the solution which has been boiled with the acid, while it 
is still hot, powdered calcium or barium carbonate until it 
is neutral. Filter and evaporate the filtrate on the steam- 
bath.1 
Dextrin. 
Dextrin is the intermediate product in the change from 
starch to glucose or maltose. There have been several 
varieties described: erythrodextrin, which is colored red 
by iodin; achroodextrin, which is not so colored, etc. 
It is formed from starch by the action of heat, acids, 
or ferments. It is soluble in water, making a sticky liquid, 
often used for a mucilage. It is produced when bread is 
toasted, and is also found in the crust. Toast or bread- 
ITo evaporate a liquid on a steam- or water- bath the 
evaporating dish in which it is contained can be heated by stand- 
ing it on a beaker of boiling water. This removes all danger of 
burning the residue. 8 
THE CARBOHYDRATES. 
crust, then, has its starch partially changed into a diffusible 
substance. 
Experiment 12.—Prepare dextrin from starch by heating 
in a porcelain dish on a sand-bath half a spoonful of powdered 
starch previously dampened with a few drops of dilute nitric- 
acid made by adding a few drops of nitric acid to a test-tube- 
ful of water. The starch must be stirred with a glass rod until 
it has turned yellowish or brown, when it has been changed to 
dextrin. 
Experiment 13.—Dissolve some dextrin in water and 
test with a drop of iodin solution. A red or brown color is 
produced, not a blue, if the change has been complete. If 
commercial dextrin is tested it will probably be found to 
contain undecomposed starch. 
Glycogen is found in a few of the lower plants, in some 
shell-fish, and in many fluids and tissues of the bodies of 
mammals. It is most abundant in the liver, and next in 
the muscles. It is also called liver-sugar or liver-starch. 
In the animal body it is most plentiful when the animal is 
well nourished, especially after a full meal. At such times 
it may be in as large an amount as 10 or 12 per cent, of 
the liver, but it is usually not more than 3 or 4 per cent. 
It disappears completely from the liver after long starva- 
tion, or more quickly through severe work or great fright. 
Glycogen. 
It is best obtained from the liver. After boiling to kill 
the ferments which are always present, dissolving in water, 
and removing the nitrogenous substances, it can be pre- 
cipitated by alcohol. 
Glycogen is an amorphous, white, tasteless powder. 
In water it dissolves to an opalescent solution. With iodin 
it gives a red color, which disappears on heating. It does GLYCOGEN. 
9 
not have a reducing action upon cupric hydrate. Boiling 
with acids converts it into dextrin, then maltose, then glu- 
cose. The salivary and pancreatic ferments produce the 
same change. 
The glycogen of the liver seems to he formed mostly 
from the carbohydrates of the food, hut partly, at least, 
from the nitrogenous compounds. 
It is deposited in the liver as a reserve material, just 
as the starch is stored for a reserve material in the plants. 
When it is needed by the body it is converted by a ferment 
into grape-sugar, and this passes into the circulation. It 
is probable that it is used to furnish energy for the body. 
After death the glycogen quickly disappears from the tis- 
sues of the body, being decomposed by the ferments which 
are present. If these are destroyed by boiling the tissue 
for a short time the glycogen is not destroyed, but can he 
extracted. 
Experiment 14.—Prepare glycogen from the liver of 
a freshly-killed, well-nourished animal. The animal is best 
killed while digestion is in progress. Remove the liver as 
soon as possible, cut it into lumps, and immediately put it 
into about four times its weight of boiling water. Let it 
boil half an hour, then rub up the lumps as much as pos- 
sible in a large mortar, add water, and boil again. Filter 
through muslin, concentrate upon the water-bath to about 
one-fourth its volume, and allow the solution to cool. Then 
precipitate the gelatin and other protein compounds by 
adding alternately small quantities of hydrochloric acid 
and potassio-mercuric iodid1 as long as anything is thrown 
1 Prepare by precipitating mercuric chloric! with potassium 
iodid, washing the precipitate and then adding it to a hot solu- 
tion of potassium iodid as long as it dissolves. 10 
THE CARBOHYDRATES. 
down. Filter and add to the filtrate twice its volume of 
alcohol to precipitate the glycogen. Wash with alcohol. 
To purify the substance it should be dissolved in a little 
water and precipitated again with alcohol. If the anhy- 
drous powder is desired, the water must be removed as far 
as possible before drying. To accomplish this wash the 
precipitate next with absolute alcohol, then with ether to 
remove the alcohol. Dry in a vacuum-desiccator over sul- 
phuric acid. If the pure substance is not desired, the tests 
may be made on the solution after the removal of the pro- 
tein compounds. 
Experiment 15.—If the dry substance has been ob- 
tained, try its taste and its solubility in water. Test the 
solution with iodin. It gives a red door. 
Experiment 16.—Try Trommer’s test. There is no 
red color if the glycogen has been purified. If it has not 
been it contains glucose, which responds to the test. 
Experiment 17.—Convert one portion of the solution 
into glucose by heating with HCI and another by the action 
of saliva. Test for the glucose by TrommeFs test. 
Experiment 18.—Prove that the glycogen is de- 
stroyed (changed to glucose) in the liver after death by the 
action of a ferment, making the test upon some liver from 
the market. Chop it finely and extract with boiling water. 
Acidify the solution slightly with acetic acid, add a little 
sodium chlorid, and boil to precipitate the protein com- 
pounds. After filtering, test the filtrate for glycogen by 
means of iodin and also for glucose by Trommer’s test. 
Experiment 19.—Add a little blood to a test-tube of 
the glycogen solution and after it has stood ten minutes 
in a beaker of water at body-temperature slightly acidify 
with acetic acid, boil, and filter to remove the albumin, and 
test the filtrate for glucose and glycogen. The latter has CELLULOSE. 
11 
been converted into glucose by a ferment which is found 
in the blood. 
Cellulose. 
Cellulose forms the membrane of the plant-cells, and 
is not found, as a constituent of the animal body, except in 
a few of the lower forms. Cotton and filter-paper are two 
of the most common examples. It is distinguished from 
the other polysaccharids by its insolubility. It is insoluble 
in the ordinary solvents, but can be dissolved in the strong 
mineral acids, being converted into dextrin. It also dis- 
solves in a solution of cupric hydrate in ammonia. 
(Schweitzer’s reagent), and in a solution of zinc chlorid 
(Schnltze’s reagent). Sulphuric acid changes paper into a 
parchment-like substance by covering the surface with a 
coating of its decomposition-products and so sticking the 
fibers together. lodin does not stain the unaltered cellu- 
lose, but does so after it has been acted upon by the acid. 
Cellulose is only slightly attacked by the digestive ferments 
of man, though the herbivorous animals digest it to a 
greater extent. By the continued action of acids it is con- 
verted into glucose. 
Experiment 30.—Show that cellulose is not stained 
hy iodin. 
Experiment 21.—Try the solubility of cotton or filter-paper 
in solution of zinc chlorid (Schultze’s reagent) and also in a solu- 
tion of cupric hydrate in ammonium hydrate (Schweitzer’s re- 
agent). It can be precipitated from these solutions by dilution 
with water. 
Experiment 33.—To one volume of water in a beaker 
add slowly two volumes of concentrated sulphuric acid, 
stirring meanwhile. Cool the mixture; then immerse in 
it for a few seconds a piece of heavy filter-paper, plunging 12 
THE CARBOHYDRATES. 
it into a large beaker of cold water as soon as it is removed. 
If tire time of immersion Iras been correct it will be semi- 
transparent after washing, and as tough as an animal mem- 
brane. It is called vegetable parchment. It can be stained 
blue by iodin. 
Experiment 23.—Let another piece of paper remain 
in a small amount of the warm acid until it has entirely 
disappeared. Then dilute a little of the acid with water 
and test it for glucose, being sure that enough alkali has 
been added to give it an alkaline reaction. 
Glucose (C6II1206). 
Glucose is also called dextrose and grape-sugar. It is 
found in the vegetable kingdom as well as in the animal. 
It is normally present in the blood and lymph and in other 
fluids of the body. Pathologically it is found in consider- 
able quantities in the urine, sometimes in as large amounts 
as 10 per cent, or more. The urine may also temporarily 
contain grape-sugar after a diet rich in carbohydrates. 
Whether it may normally occur in very small amounts in 
the urine is a question which is often discussed, but upon 
which there is no general agreement. 
Glucose is made commercially by boiling starch with 
a dilute acid. It can be produced from any of the poly- 
saccharids or disaccharids in the same manner. They unite 
with one or more molecules of water, forming glucose:— 
(CcHloos)x+ x(H2O) = x(COHI2O6). 
Cl 2H22011 -(- H2O = 2CgH1206. 
Pure glucose can be made from pure cane-sugar by 
dissolving it in alcohol and adding hydrochloric acid. The 
glucose crystallizes out on standing. GLUCOSE. 
13 
Glucose is a crystalline substance, but crystallizes with 
difficulty from water. It can better be crystallized from 
methyl alcohol or ethyl alcohol. Its taste is sweet, but less 
so than that of cane-sugar. It is easily soluble in water or 
hot alcohol. With yeast, glucose ferments best at about 
25° C., forming alcohol and carbon dioxid:— 
CgH1206 = 2C2H50H + 2C02. 
In the presence of milk or cheese it ferments to lactic 
acid. Calcium carbonate or oxid of zinc must be added to 
keep the solution neutral if it is desired that the action go 
on for a long time, as the presence of the acid kills the 
ferment:— 
C 6H1206 = 2C8HeO,. 
By the action of another ferment the lactic acid is 
changed into butyric acid:—- 
2C3Hg03 = C 4Hs02 + 2C02 -f 4H. 
Experiment 24.—Prepare pure glucose from cane-sugar by 
tlie following method: 
Acidify 100 cubic centimeters of 90-per-cent, alcohol with 
4 cubic centimeters of concentrated hydrochloric acid, warm the 
liquid upon the water-bath to 45°, and add gradually 30 grammes 
of finely-powdered cane-sugar, stirring until it has dissolved. The 
temperature should not rise above 50°. After two hours at 50° 
the sucrose has been inverted. Then let it stand in a cool place. 
The glucose commences to crystallize out in about a week, but 
crystallization may be hastened by adding to the cold solution a 
few crystals of glucose and by frequent stirring. After the glucose 
has crystallized from the solution filter, best with the aid of a 
filter-pump; wash free from the acid by 90-per-cent, alcohol, then 
by absolute alcohol; finally dry the crystals. It may be purified 
by dissolving in pure methyl alcohol by the aid of heat and 
allowing it to again crystallize out. THE CARBOHYDRATES. 
Experiment 25.—Prove that cupric hydrate (made 
by the addition of a few drops of copper sulphate to a 
sodium hydrate solution) is soluble in a solution of glucose, 
giving a deep-blue liquid. Also show that this blue solu- 
tion of copper and sugar is decomposed by heating, and 
yellow or red precipitate of cuprous oxid is produced. This 
is Trommer’s test for grape-sugar. 
Experiment 26.—Show that cupric hydrate is also 
soluble in a solution of Rochelle salt or glycerin in water, 
but that these solutions are not decomposed by boiling. 
Add to each of them a little grape-sugar and heat. Cuprous 
oxid is formed in both cases. The former is called Fell- 
ling’s test, the latter Haines’s test for glucose. 
Experiment 27.—Show that a glucose solution will 
reduce copper acetate acidified with acetic acid1 when 
heated for some time in a water-bath. This is Barfoed’s 
test. Observe the difference between glucose and lactose 
with this test. 
Experiment 28.—Prove that when heated alone in 
water cupric hydrate gives the black cupric oxid and not 
the red cuprous oxid; also that with an excess of the cop- 
per solution the black may hide the red oxid if only a small 
amount of sugar is present. 
Experiment 29.—Show that glucose will also reduce 
the subnitrate or basic nitrate of bismuth if the solution 
is made alkaline by sod ium hydrate or carbonate and boiled. 
The bismuth oxid which is formed is a black powder, but 
if mixed with much of the unreduced bismuth subnitrate 
it may appear gray. This is Boettger’s test. The bismuth 
subnitrate, like cupric hydrate, is soluble in an alkaline 
1 Reagent must contain 0.5 per cent, of acetic acid and from 
0.5 per cent, to 4.0 per cent, of copper acetate. GLUCOSE TESTS. 
15 
solution of Eoclielle salt, and this solution when heated 
with glucose gives the black oxid as a precipitate (Ny- 
landers test). 
Heat a little of the bismuth subnitrate in a solution of 
albumin which has been made strongly alkaline with sodium 
hydrate, and notice that the sulphid of bismuth, which is formed, 
has the same appearance as the black oxid which is produced by 
the glucose; that is, albumin gives a similar result to that obtained 
with grape-sugar. 
Experiment 30.—Dissolve in a small amount of water 
as much phenyl-hydrazin hydrochlorid as can be taken up 
on the point of a knife and twice as much sodium acetate, 
filtering if it is not clear. Add it to half a test-tubeful of 
the sugar solution, place the tube in a beaker of boiling 
water, and heat it for an hour. Then cool it and examine 
the precipitate with the microscope. It is phenyl-glucosa- 
zon: bright-yellow, needle-shaped crystals. They may be 
single, but are more often in clusters (Plate I, 5). They 
can be distinguished, if necessary, from similar compounds 
of the other sugars by their melting-point, which is 204° C. 
If they separate in the amorphous state, they may be crystal- 
lized, after filtering, by dissolving in a little hot alcohol, 
then evaporating the alcohol to a small volume, and letting 
it stand. 
Experiment 31.—Crush a piece of condensed yeast as 
large as a pea in a test-tube of water, and wash it two or 
three times by decantation to remove any fermentable sub- 
stances which may be present. Fill the tube completely 
full of a glucose solution. Mix and place it, still full of the 
liquid, with the mouth downward in a beaker which con- 
taius a little water, or, better, some of the grape-sugar solu- 
tion. Let it stand for twenty-four hours in a warm place. 
The carbon dioxid, which is formed, is found in the test- 16 
THE (JAEBOHYBEATES. 
tube and the alcohol in the liquid. The gas may he proved 
to he carbon dioxid by shaking it with lime-water, which 
it turns white. The presence of the alcohol is shown by 
warming the liquid after the addition of sodium hydrate 
and a little iodin. lodoform separates out in yellow scales, 
or, if the amount of alcohol is very small, the odor alone 
may be perceived. 
QUANTITATIVE TEST EOE GLUCOSE. 
Fehling’s Method.—The solutions used are: (.1) 31.64 
grammes of cupric sulphate (CuSO4, 5H20), dissolved in 
enough water to make the volume 500 cubic centimeters. 
The crystals used must be dark blue and not effloresced; 
(B) 187 grammes of pure Rochelle salt and 68 grammes of 
sodium hydrate in water enough to make the volume 500 
cubic centimeters. These solutions must be kept separate. 
Experiment 32.—For each determination mix 5 cubic 
centimeters of A with 5 cubic centimeters of B, measuring 
carefully with a pipette. Add about 40 cubic centimeters 
of water, and heat to boiling in a beaker or porcelain dish. 
If the solution is good there will be no red precipitate. 
The best results are obtained when the solution con- 
tains from 0.5 per cent, to 1.0 per cent, of sugar; that is, 
when from 5 to 10 cubic centimeters are necessary to 
destroy the blue color of the Folding solution. If it con- 
tains more than this, it must be diluted with water to 5 or 
10 times its volume, measuring accurately the water added 
and mixing thoroughly. 
The Fehling solution after dilution is heated to boil- 
ing, and the.sugar solution run in from a burette until the 
blue color has been destroyed, leaving the liquid colorless 
above the red precipitate. If too much sugar has been 
added it begins to turn yellowish. The amount of sugar LACTOSE. 
17 
is ascertained most quickly by making two determinations: 
first, a rough one, then one which is made more carefully. 
Make the first by running in the sugar solution, 2 or 3 
cubic centimeters at a time, as long as the blue color is 
well marked, then 1 cubic centimeter at a time, heating to 
boiling after each addition. It can be learned by this first 
test within 1 or 3 cubic centimeters how much will be re- 
quired. Then rinse out the beaker, take again 10 cubic 
centimeters of the Fehling solution, diluted as before; heat 
to boiling and run in at once within 1 cubic centimeter of 
the necessary amount of the sugar solution. Bring it to a 
boil. Then add the sugar solution a few drops at a time, 
heating after each addition, until the blue color has just 
been decolorized. 
Since 10 cubic centimeters of the Fehling solution is 
decolorized by 0.05 gramme of glucose, the amount of the 
sugar solution or urine which has been used from the 
burette must have contained 0.05 gramme of glucose. Bead 
the volume which has been poured from the burette, and 
calculate the percentage of sugar in the original solution. 
If this has been diluted with water the amount in the dilute 
solution must be multiplied by the number of times it was 
diluted. Bemember that however much of the sugar solu- 
tion may have been used to destroy the blue color, it con- 
tained 0.05 gramme of sugar. For example, if the amount 
used was 10 cubic centimeters, there would be 0.005 
gramme of glucose in 1 cubic centimeter; that is, in 1 
gramme of solution. In 100 grammes there would be 0.5 
gramme of glucose, or 0.5 per cent. 
Lactose is found in the milk of all mammals and occa- 
Lactose (Milk-sugar: -f II20). 
sionally during pregnancy in the urine. It can be obtained 18 
THE CARBOHYDRATES. 
from the milk by crystallization after the removal of the 
nitrogenous constituents. 
It is a crystalline substance, soluble in water, with a 
faint sweetish taste. With pure yeast it does not ferment. 
By the action of certain other ferments, however, it under- 
goes alcoholic fermentation, with the production at the 
same time of lactic acid, forming the drinks known as 
“koumiss,” when made from mares’ milk, and “kephyr” 
when from cows’ milk. The ordinary souring of milk is 
due to the formation of lactic acid from the lactose by 
micro-organisms. 
Milk-sugar gives with many reagents the same results 
as glucose. It can be distinguished from glucose by its not 
fermenting with yeast and by its having a less strong power 
of reduction. It is unable to reduce cupric compounds to 
cuprous in acetic acid solutions (Barfoed’s test). 
Experiment 33.—Preparation of milk-sugar. Dilute 
300 cubic centimeters of milk with 800 cubic centimeters 
of water, and add very cautiously not more than 0.1 per 
ceut. of acetic acid to precipitate the casein (when enough 
has been added the liquid is nearly clear). Filter. Boil the 
filtrate and filter off the coagulated albumin. Evaporate 
the filtrate upon a water-bath to a syrup and allow it to 
stand until the sugar has crystallized out. It may be puri- 
fied by recrystallizing it. 
Experiment 34.—Test the milk-sugar with Trom- 
mer’s, Fehling’s, and the phenyl-hydrazin tests, and notice 
that the results are similar to those obtained with glucose. 
Experiment 35.—Try Barfoed’s and the fermentation 
tests as made with the glucose, and observe that the results 
are negative. 
Experiment 36.—Boil the milk-sugar solution with 
a little hydrochloric acid, neutralize, and try the fermenta- SUCROSE. MALTOSE. 
19 
tion or Barfoed’s test. The lactose has been changed to 
glucose and this will now ferment, forming carbon dioxid 
and alcohol. 
Sucrose (Cane-sugar: Cl 2H22G11). 
Cane-sugar is found in plants, not in the animal king- 
dom. It has no reducing power and does not respond to 
the tests where such a reducing action occurs, such as Trom- 
mer’s, Fehling’s, and BoettgeEs. It is decomposed by heat- 
ing with acid into a molecule of glucose and one of fructose. 
Experiment 37.—Apply Trommer’s test to a solution 
of cane-sugar. It gives no results. 
Experiment 38.—80il a solution of cane-sugar with 
a little sulphuric or hydrochloric acid, neutralize the solu- 
tion, and prove that it contains glucose. 
Experiment 39.—The “invert-sugar” which results from the 
decomposition of cane-sugar by acids can be separated into glu- 
cose and fructose by adding to 10 parts 6 parts of calcium hydrate 
and 50 parts of water. Both sugars form calcium compounds. 
That with glucose, being liquid, can be pressed out of the fruit- 
sugar compound, which can then be decomposed, the fruit-sugar 
being set free, by the addition of oxalic acid as long as a pre- 
cipitate is produced. Filter and obtain the fructose by the evap- 
oration of the filtrate. 
Maltose (Malt-sugar: Cl 2H220Ix, H2O). 
Experiment 40. Preparation of Pure Maltose.—One 
hundred grammes of starch are to be mixed with 500 cubic centi- 
meters of cold water as thoroughly as possible, then heated on a 
water-bath until it makes a paste. Make a watery extract of 
malt at 40° C. from 0 or 7 grammes of dry malt. When the 
starch-paste has cooled down to 60° or 70°, add the malt-extract 
and keep it at this temperature for an hour. When the starch has 20 
THE FATS. 
been converted to maltose the liquid becomes thin and watery. 
Then boil, filter, and evaporate to a syrup upon the water-bath. 
Dissolve the maltose from the residue with small portions of 90-per- 
cent. alcohol. Distill the alcohol off from this solution, and evapo- 
rate to a syrup. Let this stand until it crystallizes. This may be 
hastened by the addition of a little crystallized maltose, which can 
be prepared by evaporating a few drops of the solution in a thin 
layer on a piece of glass. It may be purified by recrystallizing from 
methyl alcohol. 
Experiment 41.—Try the test with phenyl-hydrazin on the 
maltose. Try also Trommer’s test. It responds to both. 
THE FATS. 
The fats occur in both plants and animals. When pure 
they are colorless, odorless, and tasteless. They are in- 
soluble in water and have a lower specific gravity. They 
dissolve in hot alcohol more easily than in cold, and are 
easily soluble in ether, gasolin, or benzol. The fats mix 
with water when the two are shaken violently together, but 
they soon separate, the fats going to the top. If, however, 
something like soap or a solution of albumin is added to 
the mixture which will form a coating around the minute 
globules of fat, they are prevented from reuniting, and 
form an emulsion,—that is, a mixture of small fat-globules 
with the liquid, not a solution. It is destroyed by anything 
which will remove the coating, the fat separating again 
from the liquid. 
The fats are composed of three elements: carbon, 
hydrogen, and oxygen. They contain a much smaller per- 
centage of oxygen than the carbohydrates, the hydrogen 
and oxygen not being in the proportion to form ivater. 
When the fats are kept at the temperature of superheated 
steam or subjected to the action of the pancreatic ferment, 
they take up water and are split into two compounds: SAPONIFICATION. 
glycerin, on the one hand, and one or more of the fatty 
acids, on the other. Thus, stearin gives 
C 3H5(C18H35 02)3 + 3 H2O = C 3HS(OH)3 + 3C17H35C02H. 
stearin water zz: glycerin + stearic acid 
They may he considered then as made up of glycerin 
and a fatty acid less water. 
This splitting up of the fat-molecule is called saponifi- 
cation. It occurs when fats become rancid. It can be also 
effected by boiling the fat with a caustic alkali. Here, in- 
stead of the free fatty acid being left, it unites with the 
alkali to form a salt. These metallic salts of a fatty acid 
are the soaps:— 
C 3HB(C18H8502) + 3KOH = C 3HS(OH)3 + 3C17H35C02K. 
stearin -f- alkali zz: glycerin -j~ soap 
The soaps of the alkalies are soluble in ivater, the 
potassium compound being hygroscopic and forming soft 
soap. The sodium compound forms a hard soap. The 
compounds of the heavy metals with the fatty acids are in- 
soluble, and can be formed by adding a solution of their 
salts to a soap solution. The lead soap or lead plaster used 
in medicine is made by heating lead oxid with one of the 
fats. The soluble soaps can be thrown down from their 
solutions by saturation with a neutral salt. If a strong 
acid is added to a soap solution the soap is decomposed, 
the metal uniting with the strong acid and the fatty acid 
being set free as an insoluble substance:— 
Cl 7H85C02K + HCI = KCI + CITH36C0 JT. 
soap acid fatty acid (stearic) 22 
THE FATS. 
These fatty acids which enter into the most of the 
animal and vegetable fats are one of the unsaturated series: 
oleic acid (C17H33C0,H); and two of the saturated series: 
stearic acid (C 1 and palmitic acid (Cir,H3ICO2H). 
Besides these acids—which constitute, by far, the larger 
part of those present in fats—there are found in some cases 
certain of the lower members of the saturated series, such 
as butyric (C3H7CO2H), capronic (CSHIICO2H), caprylic 
(C7HISCO2H), and capric (C9HI9CO2H), which occur in 
butter. 
The acids which enter into the fats resemble the latter 
in many of their properties. They differ from them in 
having a slight acid reaction, the fats being neutral. They 
can also he distinguished by their not giving the irritating 
odor of acrolein as the fats do when they are heated. This 
is produced by the decomposition of glycerin, either in a 
fat or when heated alone. It is most easily obtained by 
adding before heating some substance which will assist in 
removing the water. The chemical change is 
C 3HS(OH)3 2H20 = C 3H40. 
The free acids can be neutralized by even weak alkalies, 
forming the soaps. 
The animal and many of the vegetable oils are true fats, 
differing only in that they are liquids at ordinal tempera- 
tures instead of solids. We must, however, distinguish be- 
tween these and the essential or volatile oils which are 
found in plants, hut which are not fats. The fats produce 
spots on paper which are not volatile and do not disappear 
on standing. The essential oils will disappear when left 
exposed to the air. The mineral oils belong to an entirely- 
different class of compounds, and do not contain oxygen. THE EATS. 
23 
The fats are named from the acid which they contain. 
Thus the compound of stearic acid is called stearin; of 
palmitic acid, palmitin; and of oleic acid, olein. Some- 
times the prefix tri is used with these names, as tristearin, 
etc. They differ principally in their melting-points, olein 
being a liquid at ordinary temperatures; palmitin and 
stearin, solids, the former melting more easily than the 
latter. In the animal body these fats are usually mixed, 
the consistence of the fat varying with the composition. 
Thus, the fat of the ox, or tallow, is a firmer solid than 
the fat of the hog, or lard, because it contains less of the 
olein. The animal oils contain more olein than stearin and 
palmitin, consequently they melt at temperatures below the 
ordinary ones, and are liquids. Human fat contains 67 
per cent, to 80 per cent, of olein. 
Experiment 42.—Try the reaction of a fresh fat, like 
lard or olive-oil, with a piece of litmus-paper. It is neutral; 
hut, if the fat has been standing some time and has become 
rancid, it may be slightly acid. 
Experiment 43.—Try the solubility of a few drops 
of olive-oil in a test-tube of water. It mixes when'shaken 
violent!}', hut soon separates at the top on standing. Add 
now a few drops of a soap solution and shake again. The 
liquid becomes milky and the fat does not separate. If 
the oil is not fresh it may be necessary to add a few drops 
of sodium carbonate to neutralize the free acid. 
Experiment 44.—Examine a drop of the emulsion so 
formed under the microscope. It will be found to consist 
of a great number of minute globules the size being smaller 
the more thoroughly the liquid is shaken. They are kept 
apart by the thin film of soap which covers each one. 
Experiment 45.—Add a small amount of hydro- 24 
TITE FATS. 
chloric acid and shake. The soap will be decomposed and 
the fat will collect at the top, as at first. 
Experiment 46.—Try the solubility of a fat in ether, 
chloroform, or gasolin, avoiding carefully the vicinity of a 
flame. It is easily soluble. 
Experiment 47.—Try the solubility of tallow or lard, first 
in cold, then in warm, alcohol. It is readily soluble in the warm 
alcohol and separates on cooling in the crystalline form. The 
crystals can be examined with the microscope. 
Experiment 48.—Show that the fats are non-volatile 
by placing a little upon paper and warming it over a flame. 
It does not disappear. 
Experiment 49.—To about 10 grammes (11 cubic 
centimeters) of olive-oil add 20 cubic centimeters of 10-per- 
cent. potassium hydrate solution. Boil the mixture, gently 
stirring, meanwhile, until the odor of the oil has largely 
disappeared and it appears homogeneous and no oil sepa- 
rates when a few drops are poured into water. This may 
require fifteen minutes to half an hour. Add water as it 
evaporates, to keep the original volume. The product is a 
mixture of potassium soap and glycerin. 
Experiment 60.—Convert a portion of the soap into 
the sodium or hard soap by adding some saturated salt 
solution and allowing it to stand until cold. It will dissolve 
on warming. 
Experiment 51.—To another portion add a calcium 
solution. A calcium soap is formed which is insoluble in 
water. It is this compound which is produced by the action 
of soap on “hard water.” Many of the heavy metals give 
similar compounds. Try it with solutions of iron, lead, 
copper, etc. 
Experiment 52.—To tbe remainder of the potassium 25 
THE EATS. 
soap solution add sulphuric acid slowly until it is plainly 
acid to test-paper. The fatty acids are set free as insoluble 
substances, the glycerin remaining in solution. .Filter out 
the acids by means of a wet filter-paper, through which they 
will not pass. Save the filtrate for the extraction of the 
glycerin. Wash out the sulphuric acid with distilled water 
until the wash-water is no longer acid, and try the reaction 
of the fatty acids with litmus-paper. They are acid to 
litmus. 
Experiment 53.—Allow the fatty acids to stand until the 
water has drained off or dry them by the aid of filter-paper. 
Heat them in a dry test-tube until they commence to decompose, 
and see if they give the irritating odor of acrolein. If the acids 
were washed clean this should not appear. Try the same test on 
any of the fats, and the odor will be given. It is best produced 
by adding before heating a little acid potassium sulphate. 
Experiment 54.—Neutralize the first filtrate from 
the acids, which contains the glycerin, hy adding a little 
powdered chalk, then evaporate the mixture to complete 
dryness on a water-bath. Extract the glycerin from the 
powdered mass by alcohol and evaporate this on the water- 
bath. If the glycerin is to be further purified this residue 
should be extracted Avith absolute alcohol and the alcoholic 
filtrate evaporated. The thick liquid is the glycerin, as is 
shown by the taste. 
Experiment 55.—Make the acrolein test upon the Avater-free 
glycerin by heating in a dry tube with some crystals of potassium 
bisulphate. The glycerin is decomposed, giving the acrolein odor, 
and showing that the odor Avhen obtained from fats is due to the 
glycerin radical, and not to the acid part. 
Experiment 56.-—Slioav that olive-oil is not a simple 
fat, but a mixture, hy cooling it to the freezing-point either 
hy natural cold or by a freezing mixture. It separates into 26 
THE LECITHINS. 
two parts: one crystalline, consisting mostly of palmitin, 
and the other olein, which remains liquid. 
The Lecithins. 
The lecithins are found in nearly all animal and vege- 
table cells. They are very abundant in the brain and 
nerves and in the yelk of eggs. They are sometimes called 
phosphorized fats. The formula of one of those most com- 
mon in the animal body is C44H,J0NPO!), and the composi- 
tion is probably 
f clBh35o2 
c 3h5 J clBh35o2 
has been substituted the group 
It is therefore a fat where for one stearic acid radicle 
■P030H C 2H4 N(CH3)3 oil 
In others, instead of the stearic acid radicle we may 
have the oleic or palmitic radicle. Like other fats, they 
can be decomposed or saponified. They then give phos- 
phoric acid, a fatty acid, and a base, cholin:— 
HO C 2H4 N(CH3)3 Oil. 
Lecithin is a soft, waxy substance, which swells in 
water to a pasty mass. This, under the microscope, has the 
form of oily drops or threads, the so-called “myelin” forms. 
It resembles nuclein in the readiness with which it unites 
with albuminous substances. It is found in the yelk of the 
egg in an unstable union with vitellin. THE LECITHINS. 
27 
Experiment 57. Preparation of Lecithin.—In the fol- 
lowing work the student should remember that ether and petro- 
leum-ether are very inflammable. 
Separate the albumin of an egg as completely as possible from 
the yelk. Place the yelk in a cylindrical, glass-stoppered bottle, 
add two or three times its volume of ether, and shake the bottle 
until they are well mixed. Allow it to stand until the ether above 
becomes clear, and then decant the latter into a distilling flask. Re- 
peat this extraction several times, when most of the coloring matter 
should have been dissolved. Mix the portions of ether and distill 
off the ether. The residue contains the lecithin mixed with fats, 
cholesterin, and coloring matters. Dissolve this in petroleum- 
ether and filter. Pour the filtrate into a separatory funnel, add 
about one-fourth its volume of 75-per-cent, alcohol, and shake. 
When the two liquids have separated draw off the alcohol, which 
contains most of the lecithin. Repeat this extraction with alcohol 
.several times and unite the alcoholic solutions. Distill off the 
remainder of the petroleum-ether from these, and let the solution 
stand several days in a cool place. A precipitate of cholesterin 
and other impurities will form, from which the solution is to be 
decanted through a filter. Boil the filtrate with a little animal 
charcoal to decolorize it, and filter. Evaporate at a temperature 
of 50° to 60° to a syrupy consistency. Cool this and dissolve in 
ether. If it does not dissolve completely, filter it. Evaporate the 
ether, when the lecithin remains nearly pure. If desired it can 
be purified further by dissolving in as small an amount as possible 
of warm absolute alcohol and placing this in a freezing mixture 
of —s° to —ls°, when the lecithin crystallizes out. It should be 
filtered in the cold. 
Experiment 58.—Place a little lecithin in water and ex- 
amine with the microscope. Notice the myelin forms. 
Experiment 59.—Warm this mixture with water and notice 
that after a time the lecithin turns brown and the reaction be- 
comes acid from decomposition. 
Experiment 60.-—Mix a little lecithin with dry, powdered 
potassium nitrate in a small porcelain crucible, and warm, at first 
gently, then, after deflagration, until the dark color has disap- 
peared. After cooling dissolve in water and test for phosphoric 28 
THE PROTEINS. 
acid by nitric acid and ammonium molybdate. At once 01 after 
■warming a yellow precipitate will appear. 
THE PROTEINS. 
Tire protein compounds constitute the greater part of 
the solid matter of the blood, muscles, nerves, and other 
organs of the animal body. The urine, tears, and perspira- 
tion, in a normal condition, never contain more than a 
trace. The proteins contain carbon, hydrogen, nitrogen, 
oxygen, and usually sulphur. A few contain phosphorus 
and a few others iron. When heated they are charred, 
giving off water, inflammable gases, and ammonia, at the 
same time emitting a strong odor similar to that of burnt 
horn or wool. Upon further ignition they leave an ash, 
though whether this was originally a part of the protein 
molecule has not been decided. They are often spoken of 
simply as the nitrogenous constituents of the body or the 
food, although not all of the nitrogenous compounds found 
there belong to this class. 
The proteins are very complex substances with a high 
molecular weight, and it is probably owing to this fact 
that they are so easily decomposed, as is seen by the putre- 
faction which sets in soon after life has ceased. To the 
large molecule, too, is probably due the inability of most 
of them to pass through a parchment or animal membrane. 
General Properties of the Proteins. 
Experiment 61.—Burn a small piece of dry albumin 
or other protein compound on a piece of porcelain or plati- 
num foil, or on a ivire. Notice that it turns black from THE PROTEINS. 
the presence of carbon. Observe the characteristic odor. 
On continued heating it will all disappear except the min- 
eral matters, or ash. 
Experiment 63.—Mix a few fragments of dry albu- 
min with an excess of powdered soda-lime and heat the 
mixture in a dry test-tube. Test the vapors which escape 
for ammonia, both by the odor and by their action, on a 
piece of red litmus-paper. The ammonia proves that the 
albumin contained nitrogen. 
Experiment 63.—Make a solution of lead hydrate 
by adding sodium hydrate to a small amount of lead 
acetate solution until the precipitate first formed has dis- 
solved. Add to it a protein compound, like albumin, and 
boil. The presence of sulphur in the protein compound 
is shown by the dark-colored lead sulphid, which it forms 
by uniting with the lead. 
Experiment 64.—Very small quantities of sulphur may be 
shown by changing it to a sulphid and testing for the latter with 
sodium nitroprussid. By this means it can be found in a single 
hair. Shut off the air from a Bunsen burner and, after turning it 
low, cover the hair with sodium carbonate and hold it on a wire 
in the middle of the flame. Allow the substance to fuse, being 
careful to keep it in the yellow flame to prevent oxidation. Then 
dissolve the mass in a few drops of water in a procelain dish. 
Add to the solution a very small crystal of sodium nitroprussid. 
The presence of sulphid is shown by the production of a purple 
or violet color, which is destroyed by an excess of the nitro- 
prussid. 
Experiment 05.—Test a solution of egg-albumin or any 
other albuminous substance to see if it will pass through a dialyzer. 
Only the peptones will pass through the membrane. The biuret 
test can be used to detect them (Experiment 66). THE PROTEINS. 
Classification of the Principal Protein Compounds. 
Albumin. 
Globulin. 
Albuminates 
Acid albumin. 
Alkali albumin. 
Albuminous 
substances 
Albumose. 
Peptones. 
Fibrin. 
Coagulated albumin. 
Proteins 
Mucin. 
Haemoglobin. 
Nucleoalbumin. 
Nuclein. 
Keratin. 
Elastin. 
Collagen. 
Gelatin. 
Etc. 
Protends 
Albuminoids 
Albuminous Substances. 
These are sometimes called proteins, though it is 
more convenient to reserve this name for the whole class, 
including also the proteids and albuminoids. They form 
the principal part of the protoplasm which is found in 
animal and plant cells. The composition of the molecule 
and even the exact formula of the different members of the 
group is uncertain. They are known to be very complex, 
those which have been most studied having several hun- 
dred atoms in a molecule. They differ somewhat from 
each other in composition, but their constituents usually 
lie within the following limits:— ALBUMINOUS SUBSTANCES. 
Substance. 
C 50.0 to 55.0 per cent. 62 per cent. 
H 6.5 to 7.3 per cent. 7 per cent. 
0 20.0 to 23.5 per cent. 23 per cent. 
S 0.3 to 2.2 per cent. 2 per cent. 
N 15.0 to 18.0 per cent. 16 per cent. 
Approximation. 
Average of Most Analyses. 
Phosphorus is sometimes found in less amounts than 
1 per cent. 
A few of the albuminous substances have been ob- 
tained in an imperfectly crystallized form, but most of 
them are amorphous. They differ in their solubilities and 
are classified largely upon this basis. The peptones will 
diffuse through an animal membrane, hut they do not pass 
through rapidly. 
The albuminous substances, like some other organic 
compounds which do not belong to this class, are thrown 
out of solution when to the solution certain neutral salts 
are added until it is saturated. Ammonium sulphate will 
precipitate all but the peptones and perhaps a few of the 
albumoses. Magnesium sulphate and sodium chlorid will 
precipitate many of them. 
When the albuminous substances are heated with 
water, many of them are coagulated, passing into an in- 
soluble modification. The temperature at which this takes 
place is called the coagulation-point. This is a different 
one for most of the different substances, and may be used 
in their identification and separation. It may vary, how- 
ever, from the presence of bther substances. It may be 
raised, prevented, or the coagulation made incomplete by 
alkalies or by some organic acids, like acetic acid. Coagu- 
lation is favored and the coagulation-point is lowered in 
the presence of neutral salts or small amounts of a mineral 32 
THE PROTEINS. 
acid. The concentration of the solution also may make 
it vary. Through coagulation the nature of albuminous 
substances is altered and they acquire other properties. By 
the action of alcohol albuminous compounds are precipi- 
tated, at first in an unaltered form; but if the alcohol is 
strong and acts for some time they are coagulated, and are 
then insoluble in water. 
Coagulation, when spoken of with respect to the 
protein compounds, must be distinguished from precipita- 
tion, which it resembles. When albumin is coagulated— 
e.g., by boiling, by mineral acids, or by the continued 
action of strong alcohol—it becomes insoluble in water. 
It may be precipitated without being coagulated by am- 
monium sulphate or by not too large an amount of alcohol 
and still retain its original properties, being soluble again 
upon the addition of water. 
Some of the albuminous compounds are coagulated by 
the action of ferments; for example, the fibrin, which is 
so formed from the blood or lymph. 
Albuminous substances are easily decomposed by the 
action of the putrefactive bacteria, the nitrogen and sul- 
phur uniting with hydrogen to form hydrogen sulphid, 
and ammonia, or, these two together, ammonium sulphid. 
Other nitrogen compounds are also formed, like the amido 
acids which contain the amido group, NT!,, such as leucin 
and tyrosin. Indol is also one of the nitrogenous putre- 
factive products. 
Many of the albuminous substances are precipitated 
by the mineral acids, but upon standing with an excess of 
the acid, or more quickly by heating, they are dissolved, 
going into acid albumins. Many will also form insoluble 
compounds with salts of the heavy metals, such as mercury, 
copper, and lead. With copper in an alkaline solution they REACTIONS OF ALBUMINOUS SUBSTANCES. 
33 
give a blue or purple color and upon boiling with an excess 
of nitric acid a yellow, which, becomes more reddish upon 
being rendered alkaline. MilloiTs reagent, which gives a 
red with all compounds containing a benzol nucleus united 
with an hydroxyl group, produces the same color with albu- 
minous compounds. 
GENERAL REACTIONS OF THE ALBUMINOUS SUBSTANCES. 
The tests may be made upon any albuminous com- 
pound; for example, egg-albumin. 
Experiment 66.—Make the solution alkaline with 
sodium hydrate and add a few drops of a dilute cupric 
sulphate solution. A blue or purple color results. An 
excess of the copper solution must be carefully avoided, as 
it may produce a blue color when no protein compound is 
present. (Biuret test.) 
Experiment 67.—Add a small quantity of concen- 
trated nitric acid to the albumin solution and heat to boil- 
ing. A yellow color is produced which becomes orange red 
when the liquid is made alkaline with sodium hydrate or 
ammonia. (Xanthoproteic reaction.) 
Experiment 68.—Make the solution of albumin acid 
with acetic acid, then add at least an equal volume of a 
saturated solution of ammonium sulphate, and beat to boil- 
ing. Most albuminous compounds are thrown down as a 
white precipitate. 
Experiment 69.—Acidify the solution with acetic 
acid and add a few drops of potassium ferrocyanid. A 
white precipitate is formed. 
The two last reagents fail in case of the peptones. THE PROTEINS. 
Classification of the Albuminous Substances According to Their most Important Reactions. 
Solubility. 
Coagulation on Heating. 
Precipitation by 
Albumins. 
In water. 
Coagulate. 
Saturation with neutral salts. 
Globulins. 
In diluted solutions of 
neutral salts. 
Coagulate. 
Neutral salts. 
tn • j f Acid. 
Albuminates | Alkaline. 
Acids or alkalies; not 
in cold water. 
Non-coagulable. 
Non-coagulable. 
Neutralizing. 
Neutralizing. 
Albumoses. 
Most in water. 
N on-coagnlable. 
Neutral salts, as (NTT4)2S04, 
etc. 
Peptones. 
Water. 
Non-coagulable. 
Not by (NH4)2S04 or neutral 
salts. 
Fibrin. 
Insoluble in water. 
Coagulates. , 
Coagulated Albumin. 
Insoluble in water. ALBUMINS. 
35 
Experiment 70.—Add an excess of Millon’s reagent 
and boil. A red color is produced with all albuminous 
compounds except antipeptones. 
Experiment 71.—Many of the albuminous compounds are 
also precipitated by (1) trichloracetic acid; (2) metaphosphoric 
acid; (3) alcohol or ether; (4) from solutions acidified with HCI 
or HN03 by potassium mercuric iodid or phosphotungstic acid; 
(5) from acetic acid solutions by picric or tannic acid; (6) from 
weakly alkaline solutions by salts of mercury and some of the 
other heavy metals. Try these. 
No reaction is characteristic in itself', therefore in test- 
ing for these bodies a number of tests should be tried. 
Albumins. 
The albumins are soluble in water. They are not pre- 
cipitated by dilute acids or allfalies. They are precipitated 
by saturation with ammonium sulphate and by some of tire 
salts of the heavy metals, such as mercuric chlorid and 
salts of copper, silver, and lead. For this reason albumin 
is successfully used as an antidote for poisoning by many 
of these metallic salts. By the action of acids it is con- 
verted into acid albumin and by the caustic alkalies into 
alkali albumin. 
There are several different varieties of albumin usually 
named from their sources, such as the serum-albumin of 
the blood; egg-albumin; and the albumin of milk, or lac- 
talbumin. We know that these are not the same sub- 
stances from their physiological action, as well as from 
some chemical and physical properties. If, for example, 
egg-albumin be injected into the circulation it passes in a 
short time into the urine. Serum-albumin injected in the 
same manner does not pass unaltered through the kidneys. THE PROTEINS. 
Serum-albumin is more easily dissolved by an excess of 
mineral acid than egg-albumin. They differ also in their 
coagulation-temperatures. 
Albumin may be obtained for the experiments by dis- 
solving the commercial dry egg-albumin or serum-albumin 
in cold water, filtering if it is not clear. From fresh eggs 
it can be prepared by separating the albumin from the 
yelk, being careful not to mix them. Then beat the albu- 
min, to break up the membranes in it, mix with twice its 
volume of water, and filter through a piece of unbleached 
muslin. 
Purified egg-albumin may be prepared by the following 
method:-—■ 
Experiment 72.—Separate the white from the yelk and cut 
it for some time with scissors or beat it up to a strong foam. 
Allow this to subside; filter or press it through clean, unsized 
muslin; mix it with an equal,volume of water, and, after the 
precipitate has subsided, filter it. Saturate the filtrate at about 
20° with very finely powdered magnesium sulphate, added in small 
portions with continual stirring. When it is saturated filter off 
the precipitated globulins and dialyze the filtrate until a portion 
gives only a faint turbidity with barium chlorid. The solution 
will increase in volume during the operation. It should be con- 
centrated in flat dishes at 40° to 50°, again dialyzed, and finally 
evaporated to dryness at the above temperature. It still contains 
1 to 3 per cent, of ash. 
Experiment 73.—To obtain purified serum-albumin the 
blood must be drawn into a dry vessel and the fibrin coagulated 
by beating. Filter this out through muslin and separate the cor- 
puscles from the serum by a centrifugal machine or by allowing 
it to stand in a tall cylinder until the corpuscles have settled. 
Horse-blood, if obtainable, is best for this purpose. Then draw off 
the clear serum with a siphon, dilute it with three volumes of a 
saturated solution of ammonium sulphate, and add gradually, 
stirring continually, finely powdered ammonium sulphate until the 
liquid is saturated. Filter off the precipitate, which contains 
serum-albumin and paraglobulin, and wash it with a saturated ALBUMINS. 
37 
solution of ammonium sulphate. It will be purer if it is dissolved 
in a small quantity of water and the precipitating and washing 
are repeated. Dissolve the precipitate while it is still moist in the 
smallest possible quantity of water and dialyze it. The para- 
globulin separates out as a white flocculent precipitate, while the 
albumin remains in solution. Filter out the globulin and wash 
it with water. It is insoluble in water, but soluble in dilute 
solutions of neutral salts. It is precipitated by neutral salts like 
ammonium sulphate, magnesium sulphate, or sodium chlorid. Its 
sodium chlorid solution coagulates at about 75°. 
If the solution of serum-albumin contains no globulin, it will 
not be made turbid by adding to a small portion magnesium sul- 
phate to saturation. In that ease it is to be carefully neutralized 
with ammonia, the salts dialyzed out, the contents of the dialyzer 
filtered, if necessary, and evaporated at 40° to a small volume. 
The solution should be precipitated by alcohol and immediately 
filtered. Press out the excess of alcohol as much as possible, and 
wash out the remainder with ether.1 Allow the ether to escape by 
stirring the albumin in a mortar. 
Experiment 74.—Try the effect of various reagents 
upon the coagulation-temperature of albumin by placing 
about 10 cubic centimeters of an albumin solution in a 
number of test-tubes, first filtering if necessary to obtain 
a clear liquid, and adding to (1) sodium chlorid; (3) 
concentrated sodium chlorid and a few drops of acetic 
acid; (3) a few drops of dilute hydrochloric acid; (4) 
sodium hydrate; (5) a few drops of a solution of sodium 
carbonate; (6) nothing. Set the test-tubes in a beaker of 
water and heat to boiling, observing which coagulate first. 
It will be found that acids and neutral salts favor coagula- 
tion and that alkalies hinder it or prevent it altogether. 
Experiment 75.—Test solutions of serum-albumin 
and egg-albumin with an excess of nitric acid without heat 
1 This must be done away from the vicinity of any light or 
fire. 38 
THE PROTEINS. 
and also by warming, and notice that the serum-albumin 
is more easily soluble. Try the same with concentrated 
hydrochloric acid. 
Experiment 76.-—Show that a solution of egg-albu- 
min forms an insoluble compound when added to solutions 
of mercury, silver, or copper. 
Globulins. 
The globulins are distinguished from all the other albu- 
minous substances by being soluble in dilute salt solutions, 
but insoluble in water. From this solution they are pre- 
cipitated by diluting freely with water or by removing the 
salt by dialysis. They can, in this manner, be separated 
from the albumins which remain in solution. The globu- 
lins are, as a rule, mostly precipitated by saturating the 
solution with neutral salts, like sodium chloric!. They are 
coagulated by heating with water. By the action of dilute 
acids they are converted into acid albumin. A common ex- 
ample is myosin, which is found in muscle. 
Experiment 77.-—Prepare myosin from lean meat by 
chopping about an ounce finely, then stirring it well with 
cold water to remove the albumin. Filter through muslin, 
and repeat the treatment with water until it is white or 
nearly so. Squeeze out most of the water and treat the 
residue with a 10-per-cent, solution of ammonium chloric!. 
For thorough extraction it should stand several hours, but 
enough for testing can be obtained by stirring for five 
minutes. Filter through muslin, then through paper. 
The filtrate contains the globulin (myosin). 
Experiment 78.—To a beaker full of pure water add 
a little of the solution. The myosin is precipitated. 
Experiment 79.—Heat some of the globulin solution. 
It is coagulated. GLOBULINS. ALBUMINATES. 
39 
Experiment 80.—To the solution of myosin add enough 
hydrochloric acid to make it contain 0.1 per cent. After it has 
stood a few hours syntonin, an acid albumin, has been formed. 
If the change is complete it does not coagulate upon heating. 
If there is a coagulum it ,is some of the unchanged myosin. In 
tit is ease filter it out and test the filtrate for the acid albumin by 
carefully neutralizing it with dilute sodium hydrate. The acid 
albumin which is formed from the globulin by the dilute acid is 
precipitated. 
Experiment 81.—Try the general tests for albuminous sub- 
stances (xanthoproteic, biuret, Millon’s). The myosin responds 
to all. 
The Albuminates. 
These are called also acid and alkali albumins. They 
are formed from the albuminous substances by the action 
of acids or alkalies. They are soluble in water which con- 
tains a small amount of acid or alkali, hut are not in neu- 
tral solution. Consequently they are precipitated when 
their solution is neutralized. They are not coagulated by 
boiling. The alkali albumin has the properties of an acid, 
giving a slight acid reaction. Similarly the acid albumin 
has a slight alkaline reaction. They are named from the 
manner in which they are produced, and not according to 
their reaction. 
ACID ALBUMIN. 
Experiment 82.—Prepare by adding dilute HCI to a 
solution of egg-albumin till it contains 0.1 per cent, of the 
acid. Allow it to stand an hour, at about body-tempera- 
ture, then filter and neutralize with very dilute sodium 
hydrate, being careful not to add an excess, as this would 
dissolve the precipitated acid albumin. Wash the precipi- 
tate in water. 
Experiment 83.—Notice that the acid albumin is 40 
THE PROTEIN'S. ' 
soluble in acids, though insoluble in water. Make a solu- 
tion in dilute HCI and boil. It is not coagulated. 
Experiment 84.—Make acid albumin by the action of 
strong HCI, PIN03, or acetic acid on serum-albumin or egg- 
albumin, warming if necessary. It is formed very quickly. 
Show that it gives the xanthoproteic reaction and biuret 
reaction, though it is not precipitated by boiling. 
ALKALI ALBUMIN. 
Experiment 85.—Prepare from a solution of albu- 
min by warming with an alkali, like sodium hydrate. 
Expebiment 86.—The solid alkali albumin can be obtained 
by adding strong sodium hydrate, drop by drop, to the white of 
an egg, stirring continually. No more must be added after it has 
become gelatinous, as it will then dissolve. Wash the solid in 
cold water, in which it is insoluble, though it is soluble without 
difficulty in warm water. 
Experiment 87.—Observe that the alkali albumin is 
soluble in dilute acids or alkalies. An excess of acid may 
precipitate it. 
Experiment 88.—Dissolve some of the solid sub- 
stance in hot water or use the solution obtained from 
Experiment 85. Add a few drops of phenolphthalein, 
which gives a red color, showing that the solution is alka- 
line. Add slowly dilute acid until the red color has just 
disappeared, when the solution will be neutral. The 
alkali albumin is precipitated. If, now, more acid is added, 
the precipitate dissolves. It is, consequently, like the acid 
albumin, precipitated by neutralizing. 
Experiment 89.—Show that alkali albumin, like acid 
albumin, is not coagulated by boiling. ALBUMOSES AND PEPTONES. 
41 
Experiment 90.—The thoroughly washed, solid alkali albu- 
min when pressed upon moist litmus-paper reddens it, and its solu- 
tion in calcium hydrate has an acid reaction, if no excess of the 
latter is present. To obtain this result, however, great care is 
necessary to free it from the alkali used in its preparation. 
Albumoses and Peptones. 
The peptones are the final products of the digestive 
action of pepsin upon the albuminous compounds. The 
albumoses are intermediate products between the albumi- 
nous compounds and the peptones. They may both be 
formed by the putrefaction of albuminous substances. 
The albumoses give the general reactions of the al- 
buminous substances, but they do not, like the former, 
coagulate on boiling. They are distinguished from the 
peptones hy giving a precipitate with nitric acid or potas- 
sium ferrocyanid acidified with acetic acid. They are also 
precipitated by saturating their solution with ammonium 
sulphate or sodium chlorid, then acidifying. They diffuse 
with difficulty through an animal membrane. 
The peptones do not give any of these reactions, hut 
respond to the general ones of albuminous compounds, espe- 
cially the biuret test, where the resulting color is a reddish 
pink. They do not coagulate on boiling, and, unlike the 
other albuminous substances, will pass through a parch- 
ment or animal membrane. They cannot be precipitated 
by ammonium sulphate or with potassium ferrocyanid. 
Tannin or alcohol precipitates them from their solutions. 
A number of different classes have been described, two 
of the principal ones being those designated by the prefix 
anti, which resist the action of ferments and are not easily 
decomposed further. The hemi compounds are more easily 
decomposed. Thus we have antialbumoses and hemialhu- 42 
THE PROTEINS. 
Moses, and antipeptones and hemipeptones. The other 
classes differ principally in their solubilities. 
In the dry state the albnmoses form an amorphous 
powder. The peptones also have an amorphous form, but 
are extremely hygroscopic, dissolving, to a resinous mass, 
in the water which they absorb from the air. Their taste 
is unpleasant. 
Experiment 91. Preparation of Albumoses.—Make a 
solution of hydrochloric acid containing 0.2 per cent. Add to it 
a few grains of pepsin and a small handful of blood-fibrin. Let it 
stand at a temperature of about 38° C. until the fibrin has dis- 
solved. Neutralize carefully with dilute sodium hydrate, heat to 
boiling, filter, concentrate the filtrate on the water-bath to a 
small volume, and precipitate the albumoses by adding sodium 
ehlorid until the liquid is saturated. The precipitate, which is a 
mixture of different albumoses, is filtered off, and to the filtrate is 
added acetic acid. This precipitates an albumose, called deu- 
teroalbumose. The filtrate from this will give another albumose 
as a precipitate upon saturation with ammonium sulphate. 
Experiment 92.—Dissolve some of the albumose in water 
and show that it is precipitated by potassium ferrocyanid in 
a solution acidified with acetic acid. 
Experiment 93.—Show that a solution of albumose in water 
is not coagulated by neutralizing, boiling, nor by the heating after 
addition of nitric acid to the solution, though the latter may pre- 
cipitate them in the cold (distinction from albumin and albumi- 
nates) . 
Experiment 94. Preparation of Peptone.—Digest blood- 
fibrin in a neutral solution with a watery extract from a chopped 
pancreatic gland at about body-temperature. If it is allowed to 
stand many hours, add a few crystals of thymol to prevent putre- 
faction. 'Boil after solution has taken place, filter, concentrate 
by boiling, and saturate while boiling with ammonium sulphate 
to precipitate the albumoses. Filter these out, first by muslin, then 
by filter-paper. The solution may be used for testing or the am- 
monium sulphate may be removed largely by evaporation and 
crystallization. The remainder can be removed by adding first FIBRIN. COAGULATED ALBUMIN. 
43 
barium hydrate, then barium carbonate, and boiling, until a por- 
tion of the filtrate gives no precipitate with barium chlorid. 
Experiment 95.—Commercial peptone may be used 
for testing. Test the solution of peptones hy adding first 
sodium hydrate, then not more than two or three drops of 
copper sulphate (biuret test). A red or pink color is pro- 
duced, which is characteristic of the peptones. If too much 
of the copper solution is added, the color is bluish. 
Experiment 96.—Place some of the solution in a 
dialyzer and leave it an hour, then test the solution out- 
side for the presence of peptones. They will be found to 
have passed through the membrane, although they do not 
dialyze rapidly. 
Experiment 97.—Show that tannic acid precipitates 
peptones in a neutral solution. 
Experiment 98.—Show that the peptones are not 
precipitated by potassium ferrocyanid acidified with acetic 
acid, as are the albnmoses if they contain none of the latter. 
Fibrin is formed as a gelatinous mass when fresh 
blood coagulates. If the blood is beaten during its coag- 
ulation the fibrin collects together into strings as elastic as 
caoutchouc, and remains so as long as it is moist. It can 
be freed from the blood coloring matter by washing with 
water or a salt solution. It is coagulated by heating with 
water. 
Fibrin. 
Coagulated Albumin. 
The albumins may be converted into the coagulated 
form by heating with water or by the continued action of 
strong alcohol. This is insoluble in water, but can be dis- 
solved by caustic alkalies or by heating with the strong 44 
THU PROTEINS. 
mineral acids, being thereby converted into alkali or acid 
albumins. 
The Peoteids. 
This class of substances is more complex than the 
albuminous substances. They can all be decomposed into 
albuminous substances, on the one hand, and, on the other, 
bodies which are not albuminous. Thus the albuminous 
compound is, in hasmoglobin, united with the hsematin 
molecule; in the nucleins, with phosphoric acid, etc. They 
can be considered, then, as unions of an albuminous sub- 
stance with some other substance. Most of them are coagu- 
lated by boiling. 
The Mucins. 
Mucins are found in some of the secretions of the body, 
especially in those of the mucous membrane and saliva, 
and also as a constituent of the tendons and umbilical cord. 
In their composition they resemble the albuminous sub- 
stances, but contain less nitrogen. Their characteristic 
property is that when boiled with a dilute mineral acid they 
are decomposed, giving two substances: an albuminous com- 
pound and a compound containing little or no nitrogen and 
having the power of reduction, as is shown by its changing 
cupric hydrate to cuprous hydrate in an alkaline solution. 
By this they can be distinguished from all similar albu- 
minous compounds. There are several kinds of mucin, 
although as yet they are not well differentiated from one 
another. 
The mucins are colloid substances, insoluble in pure 
water, but soluble in small amounts of dilute alkalies, such 
as calcium hydrate. They are mucilaginous and can be 
drawn out into threads. They are precipitated by the 
addition of acetic acid, if neutral salts are absent. They 45 
MUCIN S. NUCLEI) ALBUMINS. 
are not coagulated by boiling, but give many of the reac- 
tions of the albumins. Like the nncleoalbumins, they are 
acid in reaction. 
There have been sometimes included with the mucins a class 
of substances which resemble them in being decomposed by acids 
into albuminous substances and substances with the power of 
reduction, but which are not precipitated by acetic acid. They 
are more properly called mucoids. Such are the pseudomucin, 
found in ovarial liquids; chondromucoid of cartilage, and a few 
others. 
Experiment 99. Preparation of Mucin.—Mince 
finely a submaxillary gland of an ox and extract it with 
water. Filter and add to the filtrate strong hydrochloric 
acid until the liquid contains 0.15 per cent, of acid, avoid- 
ing an excess. The mucin is at first precipitated, but dis- 
solves again upon stirring. Then add two or three volumes 
of water, which will precipitate it. Separate it from the 
liquid by filtration or decantation and repeat the dissolving 
and precipitation as before. Wash with water and, if the 
dry substance is desired, with alcohol and ether. 
Experiment 100.-—Try the solubility in calcium hy- 
drate, and precipitate from this solution by acetic acid. 
Experiment 101.—Boil mucin for some time with 
dilute hydrochloric acid, and, after making the liquid alka- 
line, show by Feb ling’s test that there is a reducing body 
present. 
Experiment 102.—Show that solutions of mucin in 
an alkali will give the biuret test. 
THE ETuCLEOALBUMINS. 
The nncleoalbumins occur widely distributed in the 
animal and vegetable kingdoms, forming one of the princi- 
pal constituents of protoplasm. They are found especially 46 
THE PROTEINS. 
in the cell;, lint sometimes in the secretions, such as the milk, 
which contains casein. Some authors, however, include 
in the class of nncleoalbmnins only those found in the cell, 
and exclude such as the casein of milk and the vitellin of 
eggs. 
Chemically they are composed of the same elements 
as albumin, but contain, in addition, phosphorus and some- 
times iron. They resemble in their properties the globu- 
lins and alkali albumins. They differ, however, in contain- 
ing phosphorus. They are also insoluble in neutral salt 
solutions, unlike the globulins, and differ from the alkali 
albumins by being decomposed by the action of gastric juice 
into an albumin, which is digested, and an indigestible 
phosphorus compound called nuclein. The nucleoalbumins 
may be considered, then, as compounds of a nuclein and an 
albuminous substance. 
The nucleoalbumins are insoluble in water or dilute 
acids. They can be dissolved in alkaline solutions. They 
have the properties of dibasic acids, as is shown by the fact 
that the solution in an alkali where not too much of the 
alkali has been used have a slight acid reaction. The acid 
reaction is also shown by their setting free the carbonic 
acid of carbonates. The nucleoalbumins can be precipi- 
tated from their alkaline solutions by acidifying. The 
acid removes the alkali with which they are united and sets 
the nueleoalbumin free as an insoluble substance. This 
may be shown in the precipitation of the casein in milk. 
The nucleoalbumins are soluble in strong acetic acid 
or an excess of hydrochloric acid, being at the same time 
decomposed, nuclein being set free and the albumin being 
converted into acid albumin. They are also decomposed 
and coagulated by suspending the free nueleoalbumin in 
water and boiling. CASEIN. 
47 
In general, being compounds of the albuminous sub- 
stances, the nucleoalbumins respond to the same tests. 
Experiment 103. Preparation of Casein.—Di- 
lute about 100 cubic centimeters of milk with 400 cubic 
centimeters of water, and precipitate the casein until the 
liquid above is nearly clear by adding acetic acid drop by 
drop, avoiding an excess. Filter and wash with water. If 
fat-free substance is desired, it must be extracted in an 
extraction-apparatus with ether. For many tests this is 
not necessary. 
Experiment 104.—Test the casein for nitrogen and 
sulphur in the same manner as albumin was tested (Ex- 
periments 63 and 63). 
Experiment 105.—Test it also for phosphorus by 
mixing about a gramme of the dry substance with equal 
parts of sodium carbonate and potassium nitrate and 
fusing in a porcelain crucible. After cooling, dissolve the 
mass in water, acidify strongly with nitric acid, and add 
ammonium molybdate. A 3TIIOW precipitate, at once or 
after warming, shows the presence of phosphoric acid. 
Experiment 106.—Make the same tests upon casein 
as were made upon alkali albumin and notice that similar 
results are obtained. 
Experiment 107.—If the remain ferment is at hand 
or can be prepared, test with it a solution of casein in lime- 
water. The casein is changed into paracasein calcium, 
which is only slightly soluble in water (cheese). 
Experiment 108.—Pulverize some freshly precipitated casein 
in a mortar with water and calcium carbonate. The casein acts 
like an acid in that it sets free the carbonic acid from the car- 
bonate. 
Experiment 109.—Make a gastric Juice by adding a 48 
THE PIIOTEINS. 
few grains of pepsin to 0.3-per-cent. HG11 and digest in it 
for some time at the temperature of the body some casein. 
The nucleoalbumin is decomposed into two substances: an 
albumin, which is dissolved, and a nuclein, a compound 
rich in phosphorus, which remains. Filter out the nuclein, 
and test the filtrate with the biuret test for the albuminous 
compound. 
The Nucleins. 
The nucleins occur partly combined with albuminous 
substances as nueleoalbumins, partly free in the nucleus of 
the cell. They are composed of phosphoric acid united 
with an albumin, or sometimes in addition with a nuclein 
base, such as adenin, guanin, xanthin, or hypoxanthin. 
Besides the elements found in the albumin they contain 
phosphorus and sometimes iron. The iron in the haemo- 
globin is probably obtained by the animal organism from 
such compounds as these. We have no absolute proof that 
the common salts of iron can be assimilated by the animal 
organism. One of the nucleins which may furnish iron to 
the animal body is haematogen, found in the yelk of eggs. 
1. Those which, when acted on by hot acids or alka- 
lies, take up water and give, as decomposition-products, 
phosphoric acid and an albumin. They have been called 
paranucleins. Such a one is contained in casein. It can 
be set free by digesting by gastric juice the albumin with 
which it is united. Ibematogen is' also of this class, and 
probably furnishes the iron for the haemoglobin of the 
young bird. 
The nucleins may be divided into two classes:— 
1 This strength of acid can be obtained by diluting concentrated 
hydrochloric acid with 150 times its volume of water. 49 
THE NUCLEINS. 
2. The second class is decomposed by dilute acids or 
alkalies, giving, besides phosphoric acid and an albumin, 
one or more of the nuclein bases, also called xanthin bases, 
xanthin, guanin, etc. This class is found principally in the 
nucleus of the cell and has been called nuclear, or nucleus, 
nucleins. 
The nucleins are not attacked by gastric juice, and 
this is used to separate them from the albuminous com- 
pounds, which can he digested by it. They are insoluble 
in water and dilute acids, hut soluble in alkalies. They 
give the biuret and Milton’s tests in consequence of the 
albumin which they contain. 
Experiment 110.—Mix 50 grammes of compressed or brewers’ 
yeast with 200 grammes of water, and allow the yeast to subside. 
Pour off the water and to the residue add 0.5-per-cent, potassium 
hydrate solution; stir, and, after waiting a few minutes to dissolve 
the nucleins, filter and acidify with hydrochloric acid. Filter out 
the precipitated nucleins, wash with HCI, then hot alcohol. Dry 
over sulphuric acid. Try solubility in acids and alkalies, also biuret 
and Millon’s tests. The residue gained by fusion with sodium car- 
bonate and potassium nitrate gives a yellmv precipitate with am- 
monium molybdate, showing the presence of phosphoric acid. 
A few of the nucleins, both animal and vegetable, contain 
iron, and these are the principal source of the iron in the animal 
body, our food-materials containing no inorganic compounds of 
iron. Hsematogen is an example of such nucleins. It is found in 
the yelk of hen’s eggs united with an albuminous substance, 
which can be split off by digestion. 
The iron in such organic combinations as nuclein does not 
respond to the ordinary chemical tests until the compound has 
been decomposed by chemical agents or other means. Reagents 
like ammonium sulphid and potassium ferrocyanid decompose 
it very slowly, whereas they act immediately upon the inorganic 
compounds of iron and not at all upon hsematin. The nucleins 
which contain iron are important from their being probably the 
origin of the animal iron compounds. 50 
THE PROTEINS. 
II.EMATOGEN. 
Experiment 111.-—Prepare from the yelk of an egg. Shake 
the yelk in a wide-mouth, glass-stoppered bottle with two or three 
times its volume of alcohol; allow it to stand and when it has 
settled pour off the alcohol. Repeat this operation twice, then 
extract in the same manner or, better, in an extraction apparatus 
with ether until the residue is white. Digest this in artificial 
gastric juice (made as in Experiment 151). The nuclein, hsemat- 
ogen, remains. 
Experiment 112.—Dissolve a portion in ammonia. Test for 
iron by ammonium sulphid. At first there is no color, but after 
a time the solution turns greenish and, in twenty-four hours, black, 
as the iron is gradually set free from the compound. In the 
same manner test with potassium ferrocyanid. The Prussian blue 
is formed slowly, differing thus from its production with the ordi- 
nary iron salts. 
Experiment 113.—Add to the nuclein some hydrochloric 
acid, then, after neutralizing with ammonia, test for iron as before. 
The acid has decomposed the nuclein, so that the tests are obtained 
immediately. 
The Albuminoids. 
The albuminoids are found in the insoluble form, 
mostly in the hones of the body or the parts which are 
used for protection. They resemble the albuminous sub- 
stances, giving many of the same products when they are 
decomposed, differing, however, in other respects. They 
are not easily attacked by the reagents which dissolve and 
decompose albuminous compounds. Only the collagen and 
its derivative, gelatin, with possibly elastin, are digestible. 
Collagen. 
This substance is found in the animal body in the 
connective and cartilaginous tissues, tendons, and bones. 
That from the bones was formerly called ossein. It is com- COLLAGEN. GELATIN. 
51 
posed of the- same chemical elements as the albumins, but 
contains a little more oxygen. It is probably an oxidation- 
product of some of the albumins. It is insoluble in water, 
but by boiling with water it is converted into gelatin or 
glue. By the action of tannic acid collagen is changed to 
a form which does not putrefy. This is the action which 
takes place when leather is tanned. 
Gelatin. 
Gelatin may be considered as the hydrate of collagen, 
as it is formed by the union of collagen with water. It 
can be changed back into collagen by heating for some 
time at 130°. It swells up in cold water and dissolves 
when the water is warmed. On allowing the solution to 
cool it gelatinizes, or becomes a semisolid. It acts in this 
respect oppositely to albumin, which is soluble in the cold, 
but becomes a solid by the action of heat. 
After it has been boiled a long time with water it is 
decomposed, and does not gelatinize on cooling, peptone 
being formed. The sulphur of gelatin is united in the 
molecule in a different manner from that of the albumin 
molecule, as is proved by its decomposition-products. 
Gelatin is decomposed by the gastric juice, giving 
products similar to those from albumin. It has been 
found, however, that it cannot take the place of the al- 
buminous materials of food, though it is of value when 
used with them. 
Gelatin does not give all the reactions of the albumi- 
nous substances, although it does give the same results with 
some of them. Like the albuminous compounds, it gives 
a purple color with the biuret test; it is precipitated by 
picric acid, by mercuric chloric! in the presence of sodium TUB PROTEINS. 
chloric! and hydrochloric acid, by tannic acid in the pres- 
ence of sodium chloric!, and by saturation with ammonium 
sulphate. On the other hand, it is not coagulated by boil- 
ing; it is not precipitated by mineral acids; and it does not 
give a brown color when warmed with an alkaline solution 
of lead, as albumin does, the sulphur being apparently too 
firmly united to be split off and form lead sulphid. It 
does not give the xanthoproteic reaction when pure. 
Experiment 113 a collagen from bone by 
dissolving out the mineral constituents with dilute hydro- 
chloric acid (HCI, 1 part; water, 8 parts) until they are 
flexible, then wash out the acid. Notice that the collagen 
is not soluble in dilute acid nor cold water. To remove all 
the albumins it may be necessary to soak awhile in 5-per- 
cent. NaOH solution, then wash again. 
Experiment 114.—Convert the collagen into gelatin 
by warming it with water. Notice that it gelatinizes upon 
cooling the solution. 
Experiment 115.—80il a portion of the solution for 
some time, and notice that it is thus decomposed, so that 
it will not form a jelly upon cooling. 
Experiment 116.—Test a portion of the gelatin solu- 
tion with the biuret test. It gives a purple color like 
albumin. 
Experiment 117.-—Show that it is precipitated by 
tannic acid in the presence of Nad. 
Experiment 118.—Show that gelatin is not precipitated by 
nitric or other mineral acids, but is by saturation with ammonium 
sulphate and also by mercuric chlorid in the presence of HCI and 
Nad. 
Experiment 119.—Show that gelatin contains sulphur by 
heating with dry sodium carbonate in the reducing flame, then 
testing with sodium nitroprussid as in the case of albumin (Ex- ELASTIN. KERATIN. 
53 
periment 64), but that it gives no black sulphid of lead when 
heated in a solution of lead acetate in an excess of sodium 
hydrate (Experiment 63). 
Elastin. 
Elastin occurs in the connective tissues,—in the cervical liga- 
ment (ligamentum nucha!) very abundantly. It differs from most 
of the proteins in containing no sulphur, except possibly some 
that is loosely combined with the molecule. In the moist state it 
is very elastic; when dry it is hard and brittle. By the action 
of the digestive ferments it is decomposed into bodies called elas- 
toses, similar to the albumoses. It gives the general reactions of 
the proteins. 
Experiment 120.—Prepare elastin from the cervical ligament 
of an ox by cutting it into thin slices and boiling it for several 
days to remove the gelatin. Boil then with 1-per-cent, potassium 
hydrate for several hours, afterward with water. Repeat the boil- 
ing with 10-per-cent, acetic acid; then let it stand twenty-four 
hours in 5-per-cent, hydrochloric acid. Wash with water, boil 
with 95-per-cent, alcohol, and extract with ether to remove the 
fat. For the complete removal of the latter more than a week may 
be required. 
Experiment 121.-—Try the tests used for sulphur in albu- 
minous substances, and see that it is not present. 
Keratin. 
The keratins are the chief constituent of the horny part of 
the epidermis, of hair, horns, nails, feathers, etc. They contain 
a large amount of sulphur,—4 or 5 per cent.,—a part of which 
is so loosely united that it is set free by boiling water. It is 
owing to this sulphur that the salts of lead, silver, and some other 
metals act as hair-dyes, the sulphur uniting with the metal to 
form a dark-colored sulphid. The keratin is not at all attacked 
by the gastric or pancreatic juices. It is decomposed when heated, 
giving the odor of burnt horn. It is insoluble in water, and gives 
the xanthoproteic and Millon’s reactions. 
Experiment 122.—Prepare keratin by boiling some horn- 
shavings with water and then digesting them in succession in a 54 
FERMENTATION. 
dilute solution of pepsin containing 0.2-per-eent. HCI, and a trypsin 
solution. Wash with water, alcohol, and ether. 
Experiment 123.—Boil keratin with sodium hydrate, filter, 
and test the filtrate for sulphur by lead acetate. It produces a 
black precipitate of lead sulphid. 
Experiment 124.—Show that keratin responds to the xantho- 
proteic and Millon’s tests. 
FERMENTATION. 
By fermentation we mean the decomposition of an 
organic substance into simpler and more stable molecules, 
the agent which causes the change being itself unaffected. 
The agents are living organisms or are formed by such 
organisms. The living ferments—such as the yeast-plant 
or bacteria—are called the organized ferments. They have 
the power of reproduction and are composed of cells. The 
non-living ferments are known as the unorganized fer- 
ments, or enzymes. They may he excreted by the organ- 
ized ferments or secreted by living cells, which latter is 
the case with the digestive ferments. They are not re- 
productive and act outside of the cell where they were 
formed. The difference between the two classes may he 
seen in the action of bakers’ yeast on a dilute cane-sugar 
solution. The yeast-cell excretes an enzyme called in- 
vertin, which acts upon the sugar, producing glucose. The 
latter is fermented by the yeast-cell, giving carbon dioxid 
and alcohol. The yeast (living ferment) may be killed by 
adding chloroform or some other protoplasmic poison to 
the liquid, but the enzyme is not affected. In this case 
glucose is produced, but no further fermentation is noticed. 
The euzymes of the animal cell exist in the cells in an 
inactive condition, called zymogens, but become active 
after standing exposed to the atmosphere or being brought 
in contact with certain chemical compounds. The enzymes 55 
FERMENTATION. 
contain nitrogen and from their properties are apparently 
identical with the proteins. They are indiffusible and 
soluble in glycerin and in water. They can he mechanic- 
ally removed, without decomposition, from their solutions 
by forming precipitates therein, to which they adhere, also 
by saturating the solutions with ammonium sulphate. A 
low temperature stops their action and they are all killed 
below 100° if moisture is present. Most enzymes act best 
at about 38° C. The ferment is not destroyed, but its 
action is stopped, by a large accumulation of its own 
products. 
The organized ferments contain albumin, fat, cellu- 
lose, and some inorganic salts. They survive a high tem- 
perature better than the enzymes, but are killed at 100° 
except certain spore forms. Moisture is necessary for them 
to act. 
As is the ease with the enzymes, a sufficient amount 
of their products stops their further action. This is the 
effect of alcohol upon the yeast-plant. 
Experiment 125.—Add a little yeast to a dilute solu- 
tion of cane-sugar in water and keep it for some time at the 
body-temperature. 
Test the solution with Trommer’s test. The copper 
compound is reduced by the glucose and lievnlose which 
have been formed from the sucrose by the invertin of the 
yeast. If allowed to stand a long time the glucose is 
changed by the yeast to alcohol and carbon dioxid. 
Experiment 126.—Stir a little compressed yeast into 
lukewarm water in a test-tube, and after it has stood a few 
minutes, add a few drops of chloroform and mix thor- 
oughly by shaking. Fill the rest of the tube now with 
dilute cane-sugar solution and let it stand inverted for 
twenty-four hours in a warm place, as in Experiment 31. 56 
FERMENTATION. 
The organized ferment (yeast) is killed by the chloroform, 
so that there is no alcoholic fermentation with a formation 
of carbon dioxid. 
Experiment 127.—Test the liquid with Trommer’s 
test. It responds to the test, showing that the enzyme 
(invertin) is not destroyed, hut has decomposed the cane- 
sugar, as before, to glucose and lasvnlose. 
Experiment 128.—Separate the mucous membrane from the 
muscular coating of a pig's stomach; chop finely and allow to 
stand several hours with two or three times its weight of dilute 
phosphoric acid (1 per cent.). Filter and to the filtrate, which 
contains the pepsin, add lime-water until the reaction is alkaline. 
The calcium phosphate which falls carries down the pepsin with it. 
Miter and dissolve the precipitate in dilute HCI. Place in a 
dialyzer, changing the water outside frequently. The acids and 
salts diffuse out, leaving the pepsin inside the dialyzer, as can be 
proved by adding it to 0.2-per-eent. HCI and seeing that it will 
digest fibrin. 
Experiment 129.—Test a part of the precipitated pepsin 
which has been obtained with the calcium phosphate for nitrogen. 
This may be done, after washing with water, by drying the pre- 
cipitate, still mixed with calcium phosphate, then mixing with 
twice as much soda-lime and testing in a dry tube. The nitrogen 
is converted into ammonia, which may be recognized by the odor 
and by its action, on red litmus-paper. 
Experiment 130.-—To a part of the dialyzed-pepsin solution 
obtained in Experiment 128 add finely powdered ammonium sul- 
phate, stirring meanwhile as long as it dissolves. The pepsin is 
precipitated like the albuminous compounds. Filter, dissolve the 
precipitate in a 0.2-per-cent. HCI solution, and show that it will 
digest fibrin. 
Experiment 131.—Collect some saliva in a test-tube, 
place the latter in a beaker of water, and raise the tempera- 
ture of the water to 65° or 70° C. Keep it at this point 
for five minutes. Then let a little of it stand a few minutes 
with a starch solution, testing afterward with Trommer’s 57 
FERMENTATION. 
test. No glucose is produced, tire ferment, ptyalin, having 
been destroyed by the heat. 
Experiment 132.—Collect a considerable quantity of 
saliva, and put it into two tubes. Quickly cool one nearly 
to freezing and warm the other to body-temperature. Add 
to these an equal amount of starch solution. Allow the 
action to proceed for five minutes, then raise them both to 
the boiling-point to stop fermentation. Determine the 
amount of sugar formed by Eehling’s quantitative test 
(Experiment 32), or, approximately, by the subnitrate of 
bismuth test (Experiment 29). There has been little or 
no fermentation in the cold liquid. 
Experiment 132a.—1n the same manner cool another 
portion of the saliva and, after ten minutes, warm to body- 
temperature and show by its decomposing starch that the 
ferment is not destined by the cold. 
Experiment 133. Preparation of Lactic Acid by Fer- 
mentation.—In 150 cubic centimeters of boiling water dissolve 30 
grammes of cane-sugar and add about 30 milligrammes of tartaric 
acid. Let the solution stand two days, then add 40 cubic centi- 
meters of sour milk and about half a gramme of old cheese. After 
the addition of 15 grammes of zinc oxid allow the mixture to stand 
ten days at a temperature of 40° to 50°, with repeated stirrings. At 
the end of that time heat to boiling, filter while hot, and allow to 
cool. Zinc lactate will crystallize out on cooling if the solution is 
sufficiently concentrated. If it is not it should be allowed to evapo- 
rate to a smaller volume. It may be purified by recrystallizing. 
The acid can be obtained by dissolving the lactate in water and 
decomposing by hydrffgen sulphid gas. Filter of! the zinc sulphid, 
evaporate the filtrate to a syrup, and after it is cold extract the 
acid by dissolving it in ether.1 When the ether has evaporated 
the acid will remain. It may be preserved for testing in the gastric 
Juice tests. 
1 Remember that the vapors of ether are extremely inflam- 
mable. 58 
FERMENTATION". 
Experiment 134. Preparation of Butyric Acid.—Make 
a mixture as for the preparation of lactic acid, or use a part of that 
mixture. Allow the fermentation to go on as before, but for three 
or four weeks. In that time bubbles of hydrogen and carbon dioxid 
appear; and, after removing the zinc, butyric acid is found in the 
ether. It can be identified by its acid reaction and characteristic 
odor. 
Experiment 135. Preparation of Kephyr.-—The prepa- 
ration of the ferment requires several days. First stir the kephyr- 
grains in a little lukewarm water and decant the latter. Then 
cover them with ten times their weight of milk which has been 
boiled and cooled. Let them stand for twenty-four hours at about 
20° C., then pour off the milk, rinse the grains with a little water, 
and repeat the digestion with milk for twenty-four hours as before. 
This should be continued until the milk has an acid reaction and 
the grains rise to the upper part of the liquid, which may require 
five or six days. After this the grains may be allowed to stand 
with a moderate quantity of milk, which becomes filled with the 
organized ferment. At the end of twenty-four hours this is filtered 
through muslin, and the filtrate added to ten or fifteen times its 
volume of previously boiled and cooled milk. The whole is poured 
into a clean strong bottle, the cork is tied in, and it is allowed to 
stand two or three days at a temperature below 15° C., when the 
fermentation is complete. The grains on the muslin can be used 
an indefinite number of times in the same manner. 
Experiment 136.-—Test the acid reaction of the kephyr by 
litmus-paper (lactic acid); also show the presence of alcohol by 
the iodoform test (Experiment 31). 
THE SALIVA. 
The saliva is a mixture of the secretions of the paro- 
tid, submaxillary, and sublingual glands with that of the 
glands of the membrane of the mouth. Its reaction is 
normally faintly alkaline. The mixed saliva is a colorless, 
more or less viscid liquid, often opalescent. On standing 
it deposits calcium carbonate as a film on the surface. Ex- 
amined microscopically it is seen to contain epithelial cells THE SALIVA. 
59 
from the membrane of the mouth, air-bubbles held by 
viscid liquid, and salivary corpuscles which resemble the 
lymph-corpuscles. Bacteria are abundant. 
The normal mixed saliva contains 
| Carbonates "j | Magnesium. 
| Chlorids ! j Calcium. 
I. Inorganic <| Sulphates [ | Potassium. 
Nitrites J I Sodium. 
Sulphocjanid of potassium. 
Albumin. 
IT. Organic Mucin. 
Ptjalin. 
Nitrites and sulpboeyanids are often absent. The 
ptyalin has the power to convert boiled starch into dextrin, 
maltose, and glucose. It is not able to penetrate the gran- 
ule of the unboiled starch, or does so very slowly, differing 
in this respect from the corresponding ferment of the pan- 
creas. Its presence can be detected by mixing the saliva 
with about ten times its volume of a solution of boiled 
starch, keeping it awhile at body-temperature, and after a 
few minutes testing for sugar. The ptyalin acts best at 
about 40°, and is, therefore, not the same ferment as the 
diastase of malt, which decomposes starch most rapidly at 
a temperature of 55°. Ptyalin is destroyed by acids, even 
as dilute as the 0.2-per-cent, hydrochloric acid of the gas- 
tric juice. It is, however, probable that it acts for some 
time in the stomach before the acid penetrates the mass 
of food in a large enough quantity to stop the fermenta- 
tion. 
The secretion is influenced by the nervous system. It 
can he increased by mechanical means, like chewing a THE SALIVA. 
pebble or a piece of rounded glass in the mouth; by 
chemical action, such as touching the tongue with a crys- 
tal of tartaric acid or filling it with the vapor of ether or 
acetic acid; or by electrical excitation. In collecting saliva 
for testing it should be acomplished without trying to 
hasten its flow by suction with the tongue, as this increases 
the amount of secretion from the mucous glands and so 
dilutes the secretion of the salivary glands. 
The composition of the saliva is changed by certain 
pathological conditions. The amount is diminished in all 
febrile conditions, also in diabetes and often in nephritis. 
It is increased by the action of some medicinal substances, 
like the mercury compounds, pilocarpine, and others; also 
by anything which causes irritation or inflammation of the 
glands. Urea has been found in it abundantly during 
nephritis. The reaction becomes acid in fevers and in 
diabetes, and this sometimes happens also after long-con- 
tinued talking. 
Experiment 137. Collect for examination some 
saliva by letting it flow into the mouth without swallowing. 
Excite the flow if necessary by taking the vapor of ether 
or acetic acid into the mouth. Notice the indication of 
mucin in the viscidity, as well as the lasting foam after 
beating it with a glass rod. 
Experiment 138.—Let a portion stand exposed to the 
air, and notice the separation of calcium carbonate as a 
white film or turbidity. 
Experiment 139.—Test a portion for potassium sul- 
phocyanid by adding to it a very dilute solution of ferric 
chlorid. A red color indicates the sulphocyanid. This is 
immediately decolorized by the addition of a few drops of 
mercuric chlorid solution. 
Experiment 140.—Test for nitrites with a few drops THE SALIVA. 
61 
of a starch solution acidified with a little dilute sulphuric 
acid and containing a small amount of potassium iodid. 
A nitrite immediately gives a blue color. 
Experiment 141.—Test for albuminous substances by 
the xanthoproteic test. 
Experiment 142.—Test for mucin by adding to the 
clear saliva acetic acid drop by drop. The mucin separates 
in white, stringy flakes. They may be washed with uniter 
and tested by the mucin reactions. 
Experiment 143. Preparation of Ptyalin.—Collect a 
large amount of saliva and acidify with phosphoric acid. Then add 
milk of lime until the liquid has a faint alkaline reaction. The 
phosphoric acid is precipitated as calcium phosphate and carries 
the ptyalin down with it. Filter and allow the water to drain 
off without washing. Place the precipitate in a beaker and add 
not more water than the original amount of the saliva. Stir it 
well and filter. This removes the ptyalin from the calcium phos- 
phate, and it goes into the filtrate. Add to the filtrate an excess 
of alcohol. A white precipitate will separate, which is ptyalin 
mixed with inorganic salts. To free it from these, dissolve in a 
little water and precipitate with absolute alcohol. Repeat this 
operation if necessary. Dry it over sulphuric acid. 
Experiment 144.—Test the aqueous solution of ptyalin. It 
is not precipitated by nitric acid like albumin, nor does it give the 
xanthoproteic reaction. It can be precipitated after a time by basic 
lead acetate, the filtrate being without action on starch. 
Experiment 145.—To some boiled starch-paste cooled 
to about body-temperature add a small quantity of the 
ptyalin solution or of saliva, and after standing at that 
temperature for a short time test it with Trommer’s test 
for glucose. The latter should be present. 
Experiment 146.—Try the same experiment with un- 
boiled starch. Glucose is not formed, or, if at all, only 
after a long time and in small amounts. 
Experiment 147.—Try the effect of dilute acids by 62 
THE GASTRIC JUICE. 
diluting 1 cubic centimeter of concentrated hydrochloric 
acid with 150 cubic centimeters of water, then adding to the 
saliva an equal volume of the dilute acid. This makes the 
acidity of the whole about the same as that of the gastric 
juice. Let the acidified saliva act on boiled starch as be- 
fore. Ho glucose is produced. 
THE GASTRIC JUICE. 
The gastric juice, secreted by the glands of the stom- 
ach, differs from the other digestive fluids in having an 
acid reaction. It is a clear, thin liquid containing, as in- 
organic constituents, principally the ehlorids and phos- 
phates of tiie alkalies and of calcium and magnesium. 
There is more hydrochloric acid than can unite with the 
bases, and this must consequently he in the free state. The 
most important of the organic substances are two enzymes, 
or unorganized ferments: pepsin and rennin. 
The acidity of the gastric juice is caused principally 
by the free hydrochloric acid, hut may be at times due 
to the acid phosphates, or to the organic acids: lactic, 
butyric, and acetic. The hydrochloric acid and acid phos- 
phates are present in the normal juice. The lactic acid 
may he found in the first stages of digestion, especially 
when the food contains much of the carbohydrates, but 
is not normally found after digestion has proceeded more 
than half an hour. The acetic and butyric acids are not 
normally present. 
The free hydrochloric acid appears to be formed from 
the ehlorids which are taken with the food. Its forma- 
tion has been explained as due to the action of disodium 
phosphate upon calcium chloric! and also to the decomposi- 
tion of sodium chloric! by a weak acid, like carbonic acid. FERMENTATION IN GASTRIC JUICE. 
There are, however, many points which have not yet been 
made sufficiently clear. A free acid is necessary for the 
digestion of the nitrogenous foods by the pepsin, and this 
is one of the offices .of the hydrochloric acid. Recent re- 
searches have indicated that one of its most important 
functions is the prevention of fermentation in the stomach. 
The mineral acids have antiseptic powers even in such dilu- 
tion as that of the hydrochloric acid in the gastric juice; 
that is, from 0.2 to 0.3 per cent. Such a solution will, 
for several days, prevent putrefaction in animal matter, 
like chopped meat, which would otherwise soon commence 
to decay. It will also destroy the bacteria of many infec- 
tious diseases, though some of these are not affected when 
in the form of spores. 
The effects of increased fermentation are seen in cer- 
tain pathological conditions where the secretion of hydro- 
chloric acid is diminished or stopped. They are especially 
noticeable in the case of food containing large quantities 
of carbohydrates. The sugar which is formed by the saliva 
may be changed by the ferments into different acids:— 
C 6H1206 = 2C,HeOB. 
glucose lactic acid 
By further fermentation the lactic acid undergoes this 
change:— 
2C3H603 = C 3H7C02H + 2C02 + 2H2. 
butyric acid 
The glucose may be fermented to alcohol:— 
C 6H1206 = 2C2H50H + 3C02 
alcohol 
and this be converted to acetic acid:— 
0,11,011 + 0, = CH8C02H + 11,0. 
acetic acid THE GASTRIC JUICE. 
The so-called “heart burn,” which is one of the accompanying 
symptoms of gastric fermentation is caused by the eructations of 
gas carrying the acids up into the throat, where they cause irrita- 
tion of the mucous membrane. The methods of treatment based 
upon administration of hydrochloric acid, creasote, or other anti- 
septic substances is not for the purpose of removing the acids, but 
to stop the fermentation. Treatment with alkaline substances like 
magnesia or sodium bicarbonate neutralizes the acids, but does not 
prevent fermentation. 
The acid phosphates may he present in the normal 
gastric juice. When present they increase the acidity of the 
juice or cause an acid reaction in the absence of free acids. 
An example is the potassium compound, KH,P04, which 
is found in the stomach after meat has been eaten. 
The pepsin can be obtained from the gastric juice or 
mucous membrane of the stomach in a number ofwwars. 
Like most of the animal ferments, it can be extracted from 
the membrane by glycerin, and this glycerin solution can 
be preserved for any length of time. If it is to be used 
immediately, water containing about 0.2-per-cent, hydro- 
chloric acid can be employed for extraction. If the dry 
substance is desired, it may be thrown down with finely- 
divided precipitates of other substances, as is the case with 
many of the ferments. 
The pepsin thus obtained is probably not the pure 
substance. It is a white or yellowish-white, amorphous 
powder or scale when dried. It is hygroscopic in the air 
and has a slightly saline or acidulous taste, with no offen- 
sive odor. It is soluble in about 100 parts of water, but 
more easily on the addition of hydrochloric acid. Pepsin 
belongs to the proteins. Its molecular composition is un- 
known. 
Pepsin is inactive in neutral or alkaline liquids, but 
in slightly acid fluids it dissolves coagulated albuminous 65 
GASTRIC DIGESTION. 
compounds, with live formation of alhumoses and peptones. 
It acts most rapidly with hydrochloric acid, though others 
may he used instead. The best strength of acid for the 
purpose varies with the hind of material to be digested from 
0.1 per cent, to 0.3 per cent, of hydrochloric acid. Pepsin 
from warm-blooded animals digests best at 38°. It is 
destroyed by heating the solution. Its action is hindered 
by the presence of the products of digestion, but if these 
are removed as fast as they are formed it will change to 
the soluble form several thousand times its weight of albu- 
minous material. 
The second ferment of the gastric juice, rennin, is 
always present in human gastric juice under normal condi- 
tions. It exists in the mucous membrane in the form of a 
zymogen, which is sometimes inactive until it is set free 
by an acid. Plence if it is extracted by water it may not 
give its characteristic reaction. This is especially true in 
the case of birds or fish. This characteristic reaction is the 
coagulation of milk or casein in a neutral or faintly-alka- 
line solution in which calcium salts are present. It does 
not give the albumin reactions when in a pure state. Ren- 
nin is more easily destroyed by heating its solution than is 
pepsin. The methods of obtaining it are similar to those 
with pepsin. 
The gastric juice acts only upon the nitrogenous con- 
stituents of the food. The albuminous substances are first 
changed into acid albumin the free acid; then this is 
decomposed by the pepsin and hydrochloric acid, forming 
albumose, which passes into peptone. Since the process is 
a continuous one, all these products may be found in the 
stomach at the same time, though in the first stages the 
acid albumin is in excess and at the last the peptones. 
The connective tissues are digested by the gastric juice, 66 
THE GASTRIC JUICE. 
though the proteolytic ferment of the pancreas does not 
dissolve them. The membranes which surround the fat- 
cells are also dissolved, setting the fat free. Thus the food 
is changed into chyme: a pulpy mass which can he readily 
penetrated hy the intestinal fluids. In the first stages of 
digestion the saliva may continue to act on the starchy 
materials, and during the first fifteen to twenty minutes 
there is a formation of lactic acid, which disappears after 
this time. Milk is coagulated, partly by the acid, partly 
hy the remain. The absorption of the peptones commences 
in the stomach, but the digestion is not completed here, the 
chyme passing through the pylorus into the intestine. 
Experiment 148. Preparation oe Pepsin.—Sepa- 
rate the mucous membrane of a pig’s stomach from the 
muscular tissue. After rinsing it with water chop it finely. 
Make a dilute solution of hydrochloric acid by the addition 
of 1 cubic centimeter of concentrated acid to 150 cubic 
centimeters of water. This will contain about 0.2 per cent, 
of the pure acid. Extract the pepsin from the chopped 
membrane by means of this dilute acid. To obtain a strong 
solution of pepsin it is best to let it stand for twenty-four 
hours in a cool place, though it will he found in the solution 
in a very short time. The rennin, which is dissolved at the 
same time, is destroyed by the pepsin on standing. Filter 
the liquid through muslin, then, if necessary, through 
paper. This solution can he used directly for digestive 
experiments or for preparing the purified pepsin. 
Experiment 149.—Purified pepsin can be obtained by pre- 
cipitating it with some substance which is thrown down as a finely- 
divided precipitate. To effect this, neutralize the hydrochloric 
acid solution of pepsin in the preceding experiment and acidify 
with phosphoric acid, or extract the pepsin from the membrane 
with water acidified slightly with phosphoric acid. Precipitate by 67 
GASTRIC DIGESTION". 
adding lime-water or milk of lime until the liquid is alkaline. 
Filter off the precipitate, which contains the pepsin mixed with 
calcium phosphate. Dissolve in water with the addition of hydro- 
chloric acid. Remove the salts by dialysis. The pepsin does not 
pass through the membrane. A purer form is obtained by repeating 
the solution in acid and precipitation with lime-water or by pre- 
cipitating with alcohol before dialyzing. If the dry substance is 
desired, the drying must be at a low temperature. This may be 
done over sulphuric acid in a vacuum. 
Experiment 150.-—To make a pepsin solution which 
may he preserved, though it is somewhat impure, chop the 
mucous membrane finely, as in the previous methods, and 
after squeezing it in a piece of cloth to remove the water as 
far as possible, cover it with two or three times its volume 
of glycerin and let it stand for a week. Filter it through 
a piece of muslin, pressing out the glycerin. This gives a 
permanent solution which can be used at any time for 
digestive experiments. 
Experiment 151.—Make an artificial gastric juice by 
preparing a 0.2-per-cent, solution of hydrochloric acid as 
in Experiment 148, and adding to tins a small quantity of 
the glycerin solution of pepsin. The solution made directly 
with the dilute acid can be used if it is fresh. To this add 
a small handful of washed fibrin. If this is not at hand, 
boiled egg-albumin may be substituted for it, though in 
this case the digestion is slower. Warm the mixture 
carefully to about body-temperature and keep it at this, 
point until the albuminous substance has dissolved. No- 
tice that the edges first become transparent, then are dis- 
solved. Too high heat will destroy the ferment. The 
fibrin should be nearly digested in half an hour. It may 
be left for several days without danger of putrefaction 
provided it contains 0.2 per cent, of the free acid. 
When the fibrin has nearly or quite disappeared boil THE GASTRIC JUICE. 
the liquid to coagulate any unchanged albuminous com- 
pound, and filter. The fdtrate contains the products of 
digestion: acid albumin, albumoses, and peptones. The 
proportion of each varies with the time of digestion. The 
amount of peptones is usually small until the pepsin has 
acted for some time. 
Experiment 153.—Precipitate the acid albumin by 
neutralizing carefully with very dilute sodium hydrate. 
Filter it out and test as in Experiment 83. 
Experiment 153.—From the filtrate precipitate the 
albumoses by saturating the solution with ammonium sul- 
phate by the aid of heat. Filter and test the precipitated 
albumoses as in Experiments 93 and 93. 
Experiment 154.—The filtrate from the albumoses 
contains the peptones. Test it according to the tests used 
in Experiments 95 and 98. 
Experiment 155. Preparation of Rennin.—Extract the 
chopped mucous membrane with 0.2-per-cent, hydrochloric acid, 
as in Experiment 148. Both rennin and pepsin go into solution. 
Stir the membrane well in the acidified water, but do not let it 
stand a very long time, as the rennin is digested in an acid 
solution by the pepsin. Neutralize carefully, add a small amount 
of magnesium carbonate, and shake well. The pepsin adheres 
to the carbonate and can be filtered out with it. Shake the 
filtrate with another portion of magnesium carbonate, filter, and 
repeat the operation until the pepsin is removed, which can be 
‘shown by the failure of an acidified portion of the filtrate to 
dissolve fibrin. The filtrate containing the rennin should co- 
agulate milk in a few minutes. 
Experiment 156.—-In order to purify the rennin precipitate 
it with basic lead acetate and filter. Wash the precipitate, then 
suspend it in water, and acidify slightly with sulphuric acid. 
Filter and to the filtrate, which contains the rennin, add a 
solution of stearin soap. The latter is decomposed by the acid, 
stearic acid being set free as an insoluble precipitate. This carries RENNIN. PEPSIN. 
69 
the rennin with it. Filter and place the precipitate, with a small 
amount of water, in a glass-stoppered funnel. Add ether, shake, 
and after the ether has separated above the watery solution draw 
off the latter. The fatty acids from the decomposed soap have 
remained in the ether, leaving the rennin dissolved in the water. 
Experiment 157.—Test the solution or a specimen 
of normal or artificial gastric juice for rennin by exactly 
neutralizing, then adding it to an equal volume of milk in 
a test-tube. Place the tube in a beaker of water at body- 
temperature. If rennin is present the casein will be coag- 
ulated in twenty or thirty minutes. 
A solution of casein may be used instead of milk, but 
in order to make it coagulate a small amount of a calcium 
salt must be added. 
Experiment 158. Valuation of Pepsin (U. S. P. Proc- 
ess).—Prepare three solutions:— 
[A) To 294 cubic centimeters of water add 6 cubic centi- 
meters of dilute (10 per cent.) HCI. [B) In 100 cubic centimeters.of 
A dissolve 0.067 gramme of the pepsin. (C) To 95 cubic centimeters 
of A at a temperature of 40° C. add 5 cubic centimeters of B. 
The resulting 100 cubic centimeters of liquid will contain 2 cubic 
centimeters of dilute hydrochloric acid, 0.00335 gramme of the 
pepsin to be tested, and 98 cubic centimeters of water. 
Immerse and keep a hen’s egg during fifteen minutes in boil- 
ing water; then remove it and place it in cold water. When it is 
cold separate the coagulated albumin and rub it through a clean 
sieve having thirty meshes to the linear inch. Reject the first 
portion passing through the sieve. ’Weigh off ten grammes of the 
second, cleaner portion, place it in a flask of a capacity of about 
200 cubic centimeters, then add one-half of solution (7, and shake 
well so as to distribute the coherent albumin evenly through the 
liquid. Then add the second half of solution C, and shake again, 
guarding against loss. Place the flask in a water-bath kept at a 
temperature of 38° to 40° C. for six hours, shaking it gently every 
fifteen minutes. At the expiration of this time the albumin should 
have disappeared, leaving at the most only a few thin, insoluble 
flakes. Trustworthy results, particularly in comparative trials, 70 
THE GASTRIC JUICE. 
will be obtained only if the temperature be strictly maintained 
between the prescribed limits, and if the contents of the flasks be 
agitated uniformly and in equal intervals of time. 
The relative proteolytic power of pepsin stronger or weaker 
than that described above may be determined by ascertaining, 
through repeated trials, how much of solution B made up to 100 
cubic centimeters with solution A will be required exactly to dis- 
solve 10 grammes of coagulated and disintegrated albumin under 
the conditions given above. 
In recent years the composition of the gastric juice and 
its variations in disease are being more and more thoroughly 
studied and the results of the observations made nse of in 
clinical work. The only obstacle to the general adoption 
of these tests is probably the difficulty of obtaining the 
fluid from the stomach. 
With a little experience, the collection of the gastric juice 
for testing can be easily accomplished. In order to excite the flow 
a test-meal should be given. This should be rather simple, and 
may consist of bread or rolls with weak tea without milk. A 
large amount of food rich in albuminous materials should be 
avoided, as the peptones resulting from its digestion interfere with 
some of the tests. With such a meal digestion is at its height in 
about an hour, and the collection should be made one to one and 
a half hours after eating. 
The apparatus for withdrawing the juice is an elastic rubber 
tube about a yard in length, having a number of small perforations 
in the end or, if a large opening, it should be on the side, to avoid 
injury to the mucous membrane by suction. The perforated end 
is passed slowly down the oesophagus until it reaches the fundus 
of the stomach, known by the resistance to its further passage. 
The flow of the juice through the syphon is best started by press- 
ure upon the stomach, the outer end of the tube being held lower 
than the stomach and over a collecting vessel. By this means the 
juice is not diluted. 
Sometimes it is recommended to start the syphon by filling 
it with water by means of a funnel, then when it is full, pinching 
the top of the tube to close it and quickly lowering it. The first GASTRIC TESTS. 
71 
part of the liquid which runs out is water and should be thrown 
away, collecting only the last part. Otherwise the amounts of 
constituents found must be corrected for the water, which dilutes 
the juice. The addition of water, however, prevents accurate quan- 
titative tests. 
Before testing the Juice it should he filtered through a 
plaited filter, keeping it covered to avoid loss of water or 
acid by evaporation. 
The tests which are usually made upon the gastric 
Juice are for;— 
1. Reaction. 
2. Acid phosphates. 
3, Hydrochloric acid 
4. Organic acids ■ 
Lactic. 
Butyric. 
Acetic. 
5. Pepsin. 
Others which are not so important, but the presence 
of which may sometimes he significant, are for;— 
1. Starch. 
2. Albuminous compounds. 
3. Rennin. 
4. Blood-coloring matters. 
Qualitative tests are valuable in the cases of:— 
1. Total acidity. 
2. Hydrochloric acid. 
3. Organic acids. 
The reaction of normal gastric Juice is, of course, acid. 
In some pathological conditions it becomes neutral or alka- 
line. Litmus-paper can be used for the test. 72 
THE GASTRIC JUICE. 
To determine tire total acidity of the juice filter it, 
keeping it covered to prevent, as much as possible, evapora- 
tion; then measure accurately 10 cubic centimeters with a 
pipette and place it in a beaker. Add to this a few drops 
of an alcoholic solution of phenolphthalein, which serves 
as an indicator to tell whether the liquid is acid or alkaline 
during the determination, being red with alkalies and color- 
less with acids. Then add slowly from a burette a solution 
containing 4 grammes of sodium hydrate to the liter, stir- 
ring continually, until the liquid is a faint pink color which 
remains on standing a few minutes. Enough of the stand- 
ard alkali has then been added to neutralize the acid sub- 
stances present. Bead off this amount from the burette. 
If no gastric juice is at hand, a solution for experimental 
purposes can be made of a mixture of the above acids after 
greatly diluting them. 
The acid phosphates may be normally present, but 
cannot perform the functions of the hydrochloric acid in 
digestion. Hence it is important to be able to detect them. 
They can be distinguished from free acids from the fact 
that they are not neutralized by calcium carbonate in the 
cold as the free acids are. When, therefore, a gastric juice 
is neutralized by adding to it finely powdered calcium car- 
bonate, like precipitated chalk (which must itself be neu- 
tral), no acid phosphates are present, but the reaction was 
due to free acids. If the color of the litmus-paper is ob- 
scured by the excess of calcium carbonate, this may be rinsed 
off with distilled water. If the reaction remains acid after 
calcium carbonate has been added, acid phosphates are pres- 
ent. Their amount can be determined by finding the total 
acidity of 10 cubic centimeters, then adding calcium car- 
bonate to 15 cubic centimeters, filtering and determining 
the acidity of 10 cubic centimeters of the filtrate. This GASTRIC TESTS. 
73 
latter is due to the phosphates. The difference between 
the two is the amount of the free acids. 
Experiment 159.—Test the reaction of the acid phos- 
phates1 to litmus-paper. 
Experiment 160.—Show that they are not neutralized 
hy calcium carbonate in the cold. 
Experiment 161.—Show that the dilute free acids 
(both HCI and lactic) can he so neutralized. 
For a short time after food has been taken hydro- 
chloric acid may he wanting, or present only in traces in 
the gastric juice, without any pathological significance, 
but in one to three hours after a meal it should be found 
in larger amounts. It is then, under normal conditions, 
about 0.1 to 0.3 per cent, of the weight of the Juice. 
The common tests for the detection of hydrochloric 
acid cannot he employed in the case of the gastric juice, 
because the soluble chlorids which are usually present will 
respond to them. Special tests are used, most of which are 
based upon the fact that certain organic coloring matters 
are changed in color by a comparatively strong mineral 
acid, like hydrochloric, even in the dilute state, hut are not 
affected hy the weaker organic acids or acid salts. While 
these methods are not absolutely accurate, they are suffi- 
ciently reliable for clinical purposes when catefully per- 
formed. 
Many methods have been proposed for determining 
the amount of hydrochloric acid. Sjoqvist’s method gives 
accurate results. The juice is evaporated with barium car- 
bonate, which contains no chlorids. The acids unite with 
hAcid sodium phosphate, NaH2P04, can he prepared hy adding 
carefully orthophosphoric acid to common sodium phosphate until 
it does not precipitate barium chlorid. An excess of the acid must 
be avoided. 74 
THE GASTRIC JUICE. 
the carbonate, forming barium salts. The dry residue is 
ignited in a crucible, when the salts of the organic acids 
are destroyed, the barium chlorid remaining unchanged. 
This is dissolved with hot water and the amount deter- 
mined by a standard solution of potassium dichromate. 
This is added until the barium is precipitated and there is 
a slight excess of the dichromate. The amount of dichro- 
mate used corresponds to the amount of barium present, 
and from this the hydrochloric acid is calculated. Toepfer’s 
method is not difficult and the results are good. 
Experiment 162.—Make some 0.2-per-cent, hydro- 
chloric solution as in Experiment 109, and apply the fol- 
lowing tests:— 
1. Add a methyl-violet solution. The color changes 
from violet to blue. 
2. Add a solution of tropasolin 00. The yellow color 
is changed to a red. 
3. Add Congo red solution. The red changes to blue. 
Test-paper can be made from this reagent by dipping 
a porous paper in the solution and drying. It can be used 
in testing for TICI like the solution. 
4. Add to a few drops of liquid an equal volume of an 
alcoholic solution containing 2 per cent, phloroglucin and 
1 per cent; vanillin. Evaporate to dryness in a porcelain 
dish on a water-bath or by carefully warming over a flame. 
A rose-red color remains. (C-iinzberg’s test.) 
5. To a few drops of the HCI solution in a porcelain 
dish add a little of an alcoholic solution of resorcin and 
sugar. Warm, and a red color appears. (Boas’s test.) 
Experiment 162 a. Toepfer’s Method for Deter- 
mining the Free and Combined HCl.—lnto each of three 
beakers (A, B, and C) measure with a pipette 5 cubic 
centimeters of gastric juice. Titrate each of them with GASTRIC TESTS. 
75 
decinormal NaOH (4 grammes to the liter), using, as in- 
dicators, in A phenolphthalein, and adding the alkali until 
the liquid is a faint red; in B 3 or 4 drops of a 1-per-cent, 
solution of alizarin sodium sulphonate in water. The 
NaOH must be added until the liquid is a pure-violet color, 
not stopping with the reddish-violet shade. The exact 
shade is very nearly that given hy the indicator to a 1-per- 
cent. solution of the common sodium phosphate. With C 
the indicator is 3 or 4 drops of a 0.5-per-cent, alcoholic 
solution of dimethyl-amido-azobenzol. HCI gives a red 
color with this, and the NaOH is added until this disap- 
pears and the color changes to yellow. 
The phenolphthalein is turned red hy all the com- 
pounds with an acid reaction; the alizarin sodium sul- 
phonate reacts with all hut the acid albumin; and the 
dimethyl-amido-azohenzol only with free HCI. The test 
of A gives the total acidity; of C the free, or uneombined, 
hydrochloric acid; and the difference between A and B 
the loosely-combined hydrochloric acid, or that which is 
in combination with the albuminous compounds. The dif- 
ference between A and B C represents the acidity due 
to the phosphates and organic acids. 
Calculate from the amounts of sodium hydrate used 
Ihe percentages of each of these, remembering that one 
cubic centimeter of the alkali contains 0.004 gramme and 
neutralizes 0.003G4 gramme of HCI. The amounts of acid 
phosphates and organic acids must be expressed as equal 
to a certain amount of HCI. 
Experiment 163. Determination op Percentage op 
Free Hydrochloric Acid (Sjoqvist’s Method).—To 10 cubic 
centimeters of filtered gastric juice in a porcelain, nickel, or— 
better—a platinum crucible add enough pure barium carbonate 
to neutralize the free acid. Evaporate with a small flame to dry- THE GASTRIC JUICE. 
ness; cautiously burn the residue and heat it to a low, red heat. 
After cooling add 10 cubic centimeters of water, pulverize the 
residue, and pour off through a filter the water containing the 
dissolved barium chlorid. Repeat with successive portions of 
water until the filtrates together amount to about 50 cubic centi- 
meters. Determine in this filtrate the amount of barium chlorid 
formed from the hydrochloric acid in the following manner: Make 
a solution of 10-per-cent, acetic acid and 10-per-cent, sodium 
acetate in water and add 3 to 4 cubic centimeters of this to the 
filtrate to be tested. Add also to the liquid one-fourth to one- 
third its volume of alcohol. The latter is to make the barium 
precipitate more completely, the former solution to prevent the 
presence of free hydrochloric acid. Add from a burette a solution 
of potassium dichromate containing 7.35 grammes per liter until 
it is in excess. This is shown by taking out a drop from time to 
time on a glass rod and putting it on a piece of tetra-methyl-para- 
phenylene diamin paper. An excess of the dichromate is denoted by 
a blue color. When a faint-blue color is produced, read from the 
burette the amount of the dichromate solution used. Calculate 
from this the amount of acid in the gastric juice. The reactions 
which occur are 
2HCI + BaC03 = BaCL + CO. + H,O 
when the acid is neutralized by the carbonate, and 
2BaCL + K2Cr207 + ELO = 2BaCrO, + 2KCI + 2HCI 
when the barium salt is precipitated by the dichromate. 
One molecule of potassium dichromate, weighing 294, pre- 
cipitates two molecules of barium chlorid containing four atoms 
of chlorin. These four ehlorin-atoms were derived from four 
molecules of hydrochloric acid weighing 4 x 36.4. Therefore, for 
every 294 parts by weight of the dichromate used, 145.6 parts by 
weight of hydrochloric acid were present. One cubic centimeter 
of the dichromate solution contains 0.00735 gramme, and is, there- 
fore, equal to 0.00364 gramme of hydrochloric acid. 
The weight in grammes of the hydrochloric acid pres- 
ent in the gastric juice in the free state or combined with 
the albuminous compounds is, therefore, obtained by multi- GASTRIC TESTS. 
77 
plying the number of cubic centimeters of dichromate used 
by 0.00364, and, taking the weight of 1 cubic centimeter 
of gastric juice as 1 gramme, the percentage can be cal- 
culated. If 10 cubic centimeters of juice were used, it is 
only necessary to multiply the weight of TTCI in this by 10 
to give the percentage. 
Experiment 164. Volumetric Determination of the 
Free Acids and Acid Phosphates.-—To 10 cubic centimeters 
of filtered gastric juicel add 5 cubic centimeters of concentrated 
calcium chlorid solution and a few drops of phenolphthalein as an 
indicator, then 1/10 normal sodium hydrate until it is neutralized, 
when the color is a faint pink. The amount of alkali used cor- 
responds to the total acidity of the juice. Then take 15 cubic centi- 
meters more of the filtered gastric juice and add about a gramme 
of finely-powdered calcium carbonate. Stir well and filter through 
a dry paper. Measure 10 cubic centimeters of the filtrate into a 
small flask and by means of a rubber bulb or aspirator (not with 
the lungs) blow air through it to remove the carbon dioxid. Then 
add 5 cubic centimeters of the calcium chlorid solution and phenol- 
phthalein and neutralize with standard sodium hydrate as before. 
Since the free acids are neutralized by the calcium carbonate, the 
sodium hydrate used in this second determination corresponds to 
the acid phosphates, and the difference between the two to the 
free acid. 
Subtract the number of cubic centimeters of alkali used in the 
last determination from that used in the first. If lactic and volatile 
acids are present and have been determined, subtract also the 
number of cubic centimeters required to neutralize them. The 
remainder has been used to neutralize the hydrochloric acid. Cal- 
culate the percentage of the latter, remembering that 1 cubic centi- 
meter of sodium hydrate equals 0.00364 gramme of hydrochloric 
acid. 
Lactic acid changes a solution of gentian violet and 
ferric chlorid to a green or greenish yellow, htone of the 
1A mixture of HCI and phosphates may be used if juice 
cannot be obtained. 78 
THE GASTRIC JUICE. 
other normal or pathological constituents of the gastric 
Juice appear to do the same or to interfere with the use of 
the above reagents in testing for the lactic acid. 
Lactic acid can also he detected in the gastric juice by 
the yellow color which it imparts to a solution of ferric 
chlorid or to the amethyst solution which is produced by 
adding ferric chlorid to carbolic acid (phenol), although 
glucose or alcohol gives a similar color. 
It is generally unnecessary to determine the quantity of 
lactic acid. If this is desired it can be done by measuring off 10 
cubic centimeters of the juice, diluting to about 100 cubic centi- 
meters and distilling off the acetic and butyric acids, which can 
be determined in the distillate. After the volatile acids have been 
removed by distillation until the liquid remaining is about 10 or 
20 cubic centimeters, the lactic acid can be dissolved from the 
residue by shaking it six times with 100 cubic centimeters of 
ether, and distilling off the ether. The lactic acid remains. Dis- 
solve in water, add a few drops of phenolphthalein as an indicator, 
and see how much decinormal sodium hydrate (4 grammes per 
liter) is necessary to neutralize it. Each cubic centimeter of the 
alkali corresponds to 0.009 gramme of lactic acid. 
Experiment 165. Arnold’s Test for Lactic Acid. 
■Use the two following solutions;— 
No. 1. Saturated alcoholic solution of gentian-violet, 
0.2 cubic centimeter. 
Distilled water, 500.0 cubic centimeters. 
No. 2. Solution of ferric chlorid (U. S. lb), 5 cubic 
Distilled water, 20 cubic centimeters 
centimeters. 
In a porcelain dish place 1 cubic centimeter of No. 1 
and add 1 drop of No. 2. A bluish-violet color results. To 
this add the filtered gastric Juice. Lactic acid changes the 
color to a green or greenish yellow. GASTRIC TESTS. 
79 
Experiment 166.—To about 10 cubic centimeters of 
a 4-per-cent, solution of carbolic acid (phenol) add a few 
drops of a solution of ferric cblorid. Then dilute with 
water until the color is amethyst or reddish-violet. Use 
this as a reagent for the detection of lactic acid. The color 
is changed to yellow by the lactic acid. 
Experiment 167.—Test 0.2-per-cent, hydrochloric 
acid in the same way. The solution becomes colorless; that 
is, hydrochloric acid gives no color of its own, hence would 
not conceal the lactic acid if both should be present. Try it. 
Experiment 168.—Test the lactic acid with a solution 
of ferric chlorid so dilute that it is scarcely colored. The 
yellow color is made stronger. 
Experiment 169.—Show that glucose or alcohol will 
give a yellow color with the above reagents. 
Butyric acid can be distinguished by its odor, which is 
that of rancid butter. It can be separated from its solution 
by shaking with ether. The acid is more readily soluble in 
ether than in water, and hence remains in the ether, and is 
perceptible when the latter evaporates. It can also be re- 
moved by distillation, as it passes off with the steam. The 
acetic acid is also volatile, and if present it distills with the 
butyric acid. If the steam is condensed the quantity of 
these volatile acids can be determined by the use of a stand- 
ard solution of sodium hydrate. Since they both have the 
same significance, indicating fermentation, it is usually 
unnecessary to separate them. 
Experiment 170.—Shake 10 cubic centimeters of 
dilute butyric acid in a test-tube with about 4 cubic centi- 
meters of ether. Pour off the ether and repeat the opera- 
tion. Allow the ether to evaporate, away from lights and 
fires, and notice the odor of the acid which remains. 
The presence of pepsin in a solution like the gastric 80 
THE GASTRIC JUICE. 
juice is best detected by trying its digestive power on fibrin. 
Unless tlie liquid contains a proper amount of acid it must 
be acidified so as to contain about 0.3 per cent, of hydro- 
chloric acid. The rapidity of digestion can be best per- 
ceived by coloring the fibrin dark red with a solution of 
carmin in ammonia. This coloring matter is insoluble in 
water, but is set free, coloring the liquid, as the fibrin dis- 
solves. The depth of color denotes the amount digested. 
The colored fibrin may be kept on hand for any length of 
time by pressing out most of the water, then preserving in 
glycerin or ether. 
Experiment 171.-—Stain to a deep red some shreds 
of washed fibrin with a solution of carmin dissolved in 
ammonia. After washing with water place these in sev- 
eral test-tubes; add, to each, specimens of natural or arti- 
ficial gastric juice, containing different amounts of pepsin, 
including one with none. If they are not sufficiently acid 
make them so. Set the tubes in a beaker of water of about 
body-temperature and notice the setting free of the color 
as the pepsin acts. 
The rennin is detected by neutralizing and testing 
with milk for the power of coagulation as in Experiment 
157. 
The test for starch and its first decomposition-product, 
dextrin, is iodin dissolved in potassium iodid. According 
to von Jaksch, neither of these remains in the stomach in 
normal digestion after the first hour, though they may be 
present as long as that when there is an excess of acid or a 
deficiency of ptyalin in the saliva. 
The albuminous substances and digestive products can 
be detected by the methods given in Experiments 152, 153, 
and 154, and by the tests described for albuminous com- 
pounds. GASTRIC TESTS. 
81 
The gastric Juice never normally contains blood, but 
this is sometimes found in it or in the vomited material in 
cases of chronic ulceration of the stomach or after poison- 
ing by the corrosive or strongly irritant poisons. The 
haemoglobin is usually decomposed and the haematin which 
results gives a dark-brown color to the Juice. It is best 
identified by the haemin test (Experiment 205). 
It is occasionally desired to test the rapidity of absorption 
from the stomach. This can he accomplished by determining how 
long a time is necessary for potassium iodid to pass from the 
stomach through the circulatory system into the saliva. About 
5 grains of the iodid is taken in a capsule or in water, in the 
latter case being careful to thoroughly rinse the mouth afterward. 
The saliva is collected and tested every two or three minutes. 
Place a little upon a paper dipped in starch-paste and dried, or 
add it to a few drops of starch solution. Then add a few drops of 
a solution of calcium hypochlorite (chlorinated lime) to set free the 
iodin, which colors the starch blue. It should appear in the saliva 
in from eight to fifteen minutes. 
The clinical tests for hydrochloric acid given above are open 
to some objections. Many are interfered with by large amounts 
of albumin and peptones. If they fail in the tests of a gastric 
juice the biuret test should be tried, and if any of these substances 
are present they should be precipitated by a 10-per-cent, solution 
of tannic acid and, after filtering, the liquid should be again tested 
for hydrochloric acid. There is also a limit to the delicacy of the 
tests, so that very minute amounts of free acid may not be de- 
lected. These are so small when they cannot be thus detected that 
the condition may be considered pathological. 
Methyl-violet is turned blue by about 1/3 of a milligramme 
of acid. Tropseolin 00 is of about the same delicacy. Congo red 
is somewhat affected by the organic acids if they are not very 
dilute. The test-paper made from this has the advantage of easy 
portability and that it can be preserved and also that something 
can be judged from the imparted color of the amount of acid 
present. With a large percentage it becomes a blue black, and 
with a small amount a lighter blue. Phloroglucin and vanillin make a sensitive reagent, it being possible to detect with it one 
milligramme of free acid in 10 cubic centimeters of juice. 
THE PAN GEE ATT 0 JUICE. 
The lactic acid test—with phenol and ferric chloric!—is also 
in some cases uncertain. When it fails, however, the acid is not 
present. Some common substances, like sugar and alcohol, give 
the same results as the acid. When it is suspected that this is 
the case, the liquid should be shaken with ether to dissolve the 
acid, and the ether, after separation from the water, be evaporated 
to dryness. Dissolve the residue in a little water and test it for 
lactic acid. Arnold’s test is more reliable. 
The results obtained from chemical testing of gastric 
juice or vomited material are often of great aid in diagnosis. 
The presence of the organic acids—lactic, butyric, or acetic 
—more than thirty minutes after taking food indicates 
fermentative action, usually due to a deficiency of hydro- 
chloric acid. After the same time a failure or excessive 
amount of hydrochloric acid can be considered pathological. 
It may be absent in acute or chronic dyspepsia and in 
chlorosis. It usually is not found, or is present in only 
small amount, in carcinoma of the stomach. With dilata- 
tion of the stomach caused by stenosis of the pylorus there 
if often an hyperacidity, more than 0.4 per cent, of the 
acid being present. 
THE PANCREATIC JUICE. 
The fluid secreted by the pancreatic gland contains 
three ferments which aid in the digestion of the food: tryp- 
sin, which decomposes the nitrogenous constituents; 
steapsin, which acts upon the fats; and amylopsin, which 
converts starch into glucose. The ferments occur in the 
gland in the form of inactive zymogens, but are changed 
to the active form a few hours after death or by the action 
of water or acids. The reaction of the juice is alkaline from TRYPSIN. 
the presence of sodium carbonate. The extract made from 
the gland by means of warm water may be acid in reaction 
from the presence of sarco-lactic acid. 
The trypsin dissolves fibrin and other albuminous sub- 
stances, but differs from pepsin in that it acts in a neutral 
or weakly alkaline medium. With trypsin the fibrin does 
not swell and become transparent before dissolving, as is 
the case with pepsin, nor is acid albumin formed as the first 
stage of digestion. The principal decomposition-products 
of fibrin by the action of trypsin are, first, a globulin1; then 
albumose and peptones; then leucin, tyrosin, and aspartic 
acid. A substance called tryptophan, which gives a red- 
dish-violet precipitate with bromine-water or chlorine, is 
also produced. Little else is known of it. The antipep- 
tones are not affected by the trypsin. The hemipeptones 
are decomposed. 
The substances produced from albuminous substances 
by the action of trypsin are:— 
1. Globulin. 
2. Albumose. 
3. Peptones (called amphopeptones). 
4. Antipeptone 
(not further changed). 
5. Hemmentone. 
Leucin, tyrosin, aspartic acid, tryptophan, etc. 
Leucin (amido-caproie acid), 
CH3(CH2)3CH(]SrH2)COOH, 
1 Some albuminous substances do not give a globulin when 
acted upon by trypsin; e.g., serum-albumin. 84 
THE PANCREATIC JUICE. 
and tyrosin (para-oxy-phenyl-alpha-amido-propionic acid), 
CgH4OHCH2CH(FH2)COOH, 
are formed in the decomposition of protein substances by 
putrefaction as well as in the normal processes of digestion, 
and hence they may be found as a result of pathological 
processes where there is a destruction of the proteins. 
Leucin crystallizes in the form of thin plates, which are 
usually, when impure, grouped together into round knobs 
or halls. These can be recognized by the aid of the micro- 
scope. (Plate 11, 12, b.) In the impure state tyrosin forms 
aggregations of crystals which resemble those of leucin, but 
when purified it appears as fine silky needles often gathered 
into sheaves or halls. (Plate 11, 12, c.) Tyrosin requires 
for its solution 2454 parts of water at 20°. Leucin can he 
dissolved in 27 parts of cold water. This affords a means 
of separating them. 
Experiment 172.—Try the digestive power of tryp- 
sin on fibrin. Prepare an artificial pancreatic juice by ex- 
tracting a finely-chopped pancreatic gland of a hog or ox 
with lukewarm water. It is better to wait an hour or 
two after killing the animal, in order to allow of the forma- 
tion of the active ferment. Filter the extract through 
cloth and add to the filtrate a little chloroform or thymol 
to prevent putrefaction, which easily occurs without an 
antiseptic. Place in the liquid some fibrin, make slightly 
alkaline with sodium carbonate, and allow it to stand at 
about body-temperature until it has all dissolved. The 
products obtained will depend upon the time. If the di- 
gestion is stopped too soon they will he largely alhumoses 
and peptones, but later there will he more of the leucin and 
tyrosin. The best results will he obtained by digesting for 
several hours, or as much as a day. PANCREATIC DIGESTION. 
85 
Filter and add to the filtrate acetic acid drop by drop 
until the reaction is barely acid. The globulin is precipi- 
tated. This may he coagulated by boiling and removed by 
filtration, then the filtrate tested for the other products. 
To a small portion add an equal volume of a saturated 
salt solution, then acidify with acetic acid. If the digestion 
had not proceeded too far there is a precipitate of albumose 
(deuteroalbumose). This can he tested by the albumose 
tests. 
Saturate another small portion of the solution with 
ammonium sulphate after acidifying slightly with sulphuric 
acid. Filter out the albnmoses and try the biuret test. 
A reddish-pink color indicates that peptones are present. 
In another portion of the fluid which has digested for 
a long time test for tryptophan by adding bromine-water 
drop by drop. A reddish-violet precipitate results. It is 
destroyed by an excess of the reagent. Tryptophan is only 
found after the breaking down of the peptone molecule. 
If this is present concentrate the remainder of the 
solution to a small bulk and precipitate the albuminous 
compounds from it by the addition of about twice the 
volume of alcohol. Filter these off and evaporate the 
alcohol, or distill it if there is a large quantity. Concen- 
trate the liquid on the water-bath to a thin syrup, then 
let it stand until the tyrosin crystallizes out. Examine tbc 
form of the crystals under the microscope. If there is 
enough of the liquid to filter, remove the tyrosin and let 
the leucin crystallize from the filtrate. If there is only 
a small amount they can both be identified with the micro- 
scope. If there is a sufficient quantity they may he tested 
by the tests given in Experiments 178-181. 
Steapsin, the second ferment of the pancreatic juice, 
splits the fats into glycerin and the fatty acid, with which THE PANCREATIC JUICE. 
it was united. In the process of digestion, as it goes on in 
the animal body, only a small part of the fat is thus de- 
composed. The acid which has been set free in this manner 
unites with the carbonate of sodium which is present in the 
intestine, forming the sodium salt (a soap), and this serves 
to emulsify the rest of the fats by surrounding the globules 
with snch a coating that they are not able to unite into a 
large mass. The sodium carbonate is not able to decompose 
the fat-molecule or to form a soap with the acid until the 
latter has been set free by the ferment, nor will it emulsify 
the fat if there is no fatty acid present, but a fine emulsion 
is produced after the decomposition, and the rest of the 
fat becomes thereby capable of being absorbed through the 
walls of the intestine. 
The decomposition of the fat-molecule is as follows: 
C 8H5(C18H>BOa)3 + 3H20 = 3C17H35C02H + CaHB(OH)3. 
stearin stearic acid glycerin 
The fat-splitting ferment is easily destroyed by acids; 
hence it may not be found in a gland which lias been kept 
until it has an acid reaction. 
Experiment 173.—Make a watery infusion of the pan- 
creatic gland, as in the trypsin digestion. If it is not al- 
ready neutral make it so. Add to it in a test-tube a few 
drops of some neutral fat, like olive-oil, and let it stand 
for half an hour in a beaker of water at about 38°, shaking 
occasionally to keep the two liquids \Vell mixed. Then 
test it with a piece of blue litmus-paper. It will turn the 
paper red from the fatty acid which has been set free. 
Experiment 174.—Shake, in a test-tube of water, to which 
a few drops of sodium carbonate have been added, a few drops of 
olive-oil which does not contain free acids. (Since the oil easily 
becomes decomposed on standing, it may be necessary to remove PANCREATIC DIGESTION. 
87 
the free acids by first shaking with very dilute sodium hydrate 
solution and ether. Separate the ether from the water, wash well 
by shaking it a number of times with pure water, and allow it 
to evaporate at a gentle heat away from a fire or lamp. The 
alkali has united with the free acid to form a soap, and this has 
been washed out by the water, leaving the fat neutral.) If the 
oil is neutral it will form no emulsion with the carbonate. Add 
now a drop of a fatty acid like oleic acid and shake. A fine 
emulsion is formed immediately. 
Experiment 175.—If a bottle of rancid oil is at hand, shake 
it with a weak solution of sodium carbonate, and notice that it 
contains enough of the free acids to form an emulsion at once. 
The third ferment of the pancreas, amylopsin, con- 
verts starch into glucose as the ptyalin does, except that 
its action is more energetic. Thus it acts upon raw starch, 
which the ptyalin will not do, or at most only slowly. 
Experiment 17G.—Treat boiled and unboiled starch 
with a small amount of the watery solution, of the pan- 
creatic ferments made as before. Test for glucose by Trom- 
mer’s test. Notice that it is found with the boiled starch 
in a few seconds; after a longer time with the unboiled. 
Experiment 177.—To prepare leucin and tyrosin in larger 
quantities, take of white horn-shavings 2 parts and boil for 
twenty-four hours with 13 parts water and 5 parts of concentrated 
sulphuric acid, adding water as it evaporates. Dilute with water, 
and while warm neutralize with milk of lime. Filter, boil the 
precipitate several times with water, and filter in order to remove 
all the leucin and tyrosin. Unite these filtrates, and, after con- 
centrating them by boiling, precipitate the calcium by the addition 
of oxalic acid without using an excess. Filter, extract the precipi- 
tate with boiling water, unite the filtrates, and evaporate until it 
becomes a thin syrup. The last of the evaporation should be per- 
formed on a water-bath, to avoid burning. Allow it to stand and 
crystallize. Tyrosin at first crystallizes out, mixed with a little 
leucin. Filter, concentrate the filtrate, and allow the leucin to 
crystallize. 
Another better way to separate the mixed substances is to 88 
TYROSIN. LEUCIISr. 
dissolve them in a large quantity of boiling water to which am- 
monia has been added. To the boiling solution add basic lead 
acetate solution until the precipitate formed is nearly white. 
Filter, heat the filtrate to boiling, neutralize with sulphuric acid, 
and filter while hot. After cooling, the tyrosin crystallizes, and 
can be filtered off, while the leucin remains in solution. Remove 
the lead from the filtrate which contains the tyrosin by passing 
hydrogen sulphid through it, filter, and concentrate the filtrate, 
then boil it with freshly-prepared copper oxyhydrate. (This is 
made by precipitating a solution of copper sulphate with sodium 
hydrate and washing by decantation until it no longer has an 
alkaline reaction.) Part of the leucin is thrown down as the copper 
salt and a part remains in solution. The precipitate can be re- 
moved by filtration and the leucin obtained in the pure state by 
removing the copper by hydrogen sulphid and allowing the leucin 
to crystallize after concentration. From the deep-blue filtrate the 
copper salt can be obtained by evaporating it to a small volume 
and letting it crystallize. It forms sky-blue aggregations of 
crystals. If desired, the copper can be removed from these also 
by hydrogen sulphid. The leucin thus obtained is not as pure as 
the first portion. 
Experiment 178.-—Test the tyrosin with Millon’s reagent. It 
gives a red color, showing the presence in the molecule of the group 
—CeipOH. 
Experiment 179.—To a portion of tyrosin crystals as large 
as half a pea add, in a porcelain dish, a few drops of concentrated 
sulphuric acid and warm on the water-bath. This forms tyrosin 
sulphuric acid. This is diluted with water and enough barium 
carbonate added to neutralize it, then filtered. The filtrate con- 
tains the tyrosin compound, which, with a very dilute solution of 
ferric chlorid, gives a deep-violet solution of tyrosin ferric sulphate. 
Experiment 180.—Evaporate a small portion of the leucin 
crystals with a drop of nitric acid upon platinum foil. The residue 
is colorless, but on adding a drop of sodium hydrate becomes yel- 
low to brown, and on gently heating rolls around on the foil in 
the form of drops. 
Experiment 181.-—Place a few crystals of leucin in a dry 
test-tube and heat gently. They melt with the odor of amylamin THE BLOOD. 
89 
and sublime, the leucin appearing on the sides of the tube as 
wooly flakes. This may not succeed if the leucin is very impure. 
THE BLOOD. 
In the examination of the blood it is convenient to 
consider it as composed of two parts: the corpuscles and 
the alhnminons liquid in which they are suspended,—the 
plasma. The plasma, on standing, separates into two 
parts by coagulation, the clot—or fibrin—and a liquid,— 
the serum. 
The reaction of the blood is alkaline from the pres- 
ence of the carbonate and phosphate of sodium. The 
specific gravity varies from 1.045 to 1.075, with an aver- 
age for adult human beings of about 1.055. 
The color of the blood is caused by the red corpus- 
cles. Even comparatively thin layers of the blood are 
opaque from their presence. The coloring matter (haemo- 
globin) can he set free from the inside of the corpuscle 
by water or h}r many chemical reagents. The color be- 
comes then much darker, since the light is no longer re- 
flected from the surface of the corpuscles. The addition 
of strong neutral salt solutions to blood turns it bright 
red, because of the increased reflection of light from the 
shriveled corpuscles. 
The red corpuscles of the same species of animals 
have the same shape. The average size of those of one 
animal of a species will be the same as that of any other, 
although the size of the individual corpuscle may vary 
greatly in the same animal. In most mammals they are 
round, biconcave, non-nucleated disks. In the blood of 
birds, amphibians, and most fishes they are nucleated and 
more or less elliptical. A single corpuscle when seen un- 
der the microscope has a yellowish color, not a red. The 90 
THE BLOOD. 
size of the red corpuscles can be greatly changed by add- 
ing water or strong solutions of neutral salts. When 
water is added it passes in by diffusion, and the corpus- 
cle swells and may burst. Likewise by diffusion when 
they are placed in a liquid and it contains more salts than 
the blood, water passes out and the corpuscle becomes 
smaller and shriveled in appearance. In order to dilute 
the blood without changing the size a solution containing 
0.5 to 0.6 per cent, of sodium chlorid can be used. 
Freshly-drawn blood, when allowed to stand undis- 
turbed, in a few minutes becomes thickened to a dark- 
red gelatinous mass. If the coagulation is slow the red 
corpuscles have time to sink and collect with the fibrin 
in a mass in the lower part of the vessel. The serum is 
squeezed out of the mass and surrounds it, above and at 
the sides. If the blood is beaten during the time of co- 
agulation the fibrin does not separate as a gelatinous 
substance, but in stringy masses, which have a high de- 
gree of elasticity. The coagulation can be prevented or 
hindered by cold and by the addition of neutral salts, 
peptones, and some other substances. 
To determine the number of red corpuscles the apparatus of 
Thoma-Zeiss may be employed. This consists of two pieces; a 
pipette for measuring and diluting the blood and a cell for count- 
ing the number with the aid of a microscope. The lower part of 
the pipette is a graduated capillary tube for measuring the blood. 
Above is a bulb which, being filled to the mark with the diluting 
fluid, dilutes 200 times the blood which was measured in the capil- 
lary tube. The counting-cell when covered with a cover-glass gives 
a layer of blood 0.1 millimeter in depth. On the bottom of the cell 
are ruled sixteen squares, each 1/m of a square millimeter in area. 
They are surrounded by two rows of smaller rectangles. The 
volume of blood over each of these squares, then, must contain 
V-tooo cubic millimeter. If the number of corpuscles which are con- 
tained in this V4ooo cubic millimeter is determined, the number in NUMBER OF CORPUSCLES. 
91 
any volume can be found by multiplication. Several different 
solutions have been proposed for the dilution of the blood, one of 
the most convenient being a 3-per-cent, solution of sodium chlorid. 
For clinical purposes the blood is best obtained from the tip of the 
finger. For testing the method defibrinated blood from the 
slaughter-house may be employed. The dilution may be made 
twice as great by filling the capillary only to the 0.5 mark and 
diluting as before. In this case the number of corpuscles in each 
square must be multiplied by 8000 to give the number in a cubic 
millimeter. 
The average number of red corpuscles normally present is 
5,000,000 per cubic millimeter in the case of a man, and 4,500,000 per 
cubic millimeter of a woman. This may vary greatly in disease. 
Experiment 182. Determination of the Number of 
Red Blood-corpuscles.—Fill the capillary tube of the pipette 
with blood to the mark 1, drawing it in slowly by suction Avith 
the mouth and avoiding the presence of air-bubbles in the tube. 
Quickly Avipe dry the end of the pipette with filter-paper and draAV 
in the diluting salt-solution (3 per cent.) to the mark 101. Close 
the loAver end of the pipette Avith the finger, then compress the 
rubber tube at the upper end of the pipette and shake to thoroughly 
mix the fluids. The small glass bead in the bulb aids in this mix- 
ing. Allow the salt solution to Aoav out of the capillary, place a 
drop of the diluted blood on the ruled side and cover it with a 
cover-glass so that air-bubbles are not inclosed. When the cor- 
puscles have come to rest, count the number in all sixteen squares. 
Count those upon the upper and left hand line of each square as 
belonging to the square. Use an objective which Avill magnify 
100 to 200 diameters. The squares should be taken in some definite 
order to avoid counting the corpuscles in the same one more than 
once. The average number of corpuscles multiplied by 4000 girms 
the number in a cubic millimetre of the diluted blood. Multiply 
this by 100 for the original blood. Since a slight error in the 
average of each square Avould make a considerable error Avhen 
multiplied by 4000, it is advisable to repeat the filling of the cell 
several times before taking the average. After using, the pipette 
should be rinsed out first with the diluting salt solution, then suc- 
cessively Avith Avater, alcohol, and ether, and finally dried by bloAV- 
ing dry air through it. 92 
THE BLOOD. 
Experiment 183.—Separation of the Corpuscles 
from the Serum.—Add 30 cubic centimeters of a satu- 
rated solution of sodium chlorid to 270 cubic centimeters 
of water, then mix with it 30 cubic centimeters of blood, 
which lias been defibrinated by beating it while freshly 
drawn. Pour it into a flat-bottomed, shallow dish and 
allow it to stand until the corpuscles have settled. Decant 
the serum, and, after mixing the corpuscles with more salt 
solution as before, allow to settle and decant again. By 
this means the serum can be entirely removed. 
Directions for obtaining the serum-albumin and 
serum-globulin have already been given (Experiment 73). 
Experiment 184. Determination of the Specific 
Gravity of Blood.—Prepare a mixture of benzol and 
chloroform, of which the specific gravity when tested with 
a sensitive hydrometer shall he somewhat less than that 
of blood. Into this mixture allow a drop of blood to fall. 
Freshly-drawn blood—for example, from the end of the 
finger—is best, though the method can be demonstrated 
by the use of defibrinated blood. When the blood has 
sunk, add chloroform, drop by drop, stirring meanwhile, 
until the drop floats in the midst of the liquid; that is, 
it has the same specific gravity. Then filter out the blood, 
covering the funnel to prevent evaporation, and determine 
the specific gravity of the mixture by means of a sensitive 
hydrometer. The mixed liquids can be preserved for 
future tests. 
Experiment 185.—Test the reaction of fresh blood, 
using a piece of neutral, glazed litmus-paper. After rins- 
ing off the blood with a little distilled water the paper is 
blue. 
Experiment 186.—T0 a little blood in a test-tube THE BLOOD. 
93 
add an equal volume of water, and notice the change in 
color. 
Experiment 187.—T0 another portion add as much 
saturated salt solution and observe that the color becomes 
brighter. 
Experiment 188.—Examine each of these under a 
microscope and compare the appearance of the corpuscles 
with those of the fresh blood. 
Experiment 189.—Place in a test-tube half an inch of a cold, 
saturated solution of sodium sulphate. Open the carotid vein of 
a rabbit or other small animal and allow twice as much blood to 
flow into the tube. Collect as much more in a clean, perfectly dry 
tube and allow both to stand twenty-four hours. In the first tube 
there is no coagulation, but the corpuscles settle toward the bot- 
tom. In the second the corpuscles are mostly held in the mass of 
coagulated fibrin from which drops of serum are pressed out. 
Experiment 190.—Separate the serum from blood 
by collecting the freshly-drawn blood in a shallow vessel 
and letting it stand covered until it has coagulated and 
the serum is pressed out by the contraction of the coagu- 
lated mass. 
Experiment 191.—Show that the serum contains 
albumins by testing a diluted solution by 
1. Heat. 
3. Biuret test. 
3. Xanthoproteic test. 
Experiment 193.—Prepare fibrin by beating freshly- 
drawn blood with a fork or a bundle of switches. When 
it has coagulated, pour off the liquid and preserve it for 
further tests. Wash the fibrin, at first in water to which 
a little salt has been added, then in clear water. Break 
up the large clots and continue the washing until the color- 
ing matter is removed. If it is desired to keep it, it can THE BLOOD. 
be preserved in a 1-per-cent, solution of corrosive subli- 
mate. 
Experiment 193.—After noticing the structure and 
elasticity of fibrin apply the following tests:— 
1. Xanthoproteic. 
2. Insolubility in hot and cold water. 
3. Swelling and gradual solution in dilute acid. 
Experiment 194.—Dissolve a little dried blood in 
nitric acid with the aid of heat. Filter and test the filtrate 
for iron with potassium ferrocyanid, which produces a blue 
color. 
Hemoglobin and its Derivatives. 
I. Hemoglobin : Composed of an albuminous substance 
and an iron compound,—hemochromogen. 
11. Oxyhemoglobin : A compound of oxygen with haemo- 
globin. 
111. Methemoglobin: Composition same as oxyhemo- 
globin. Different arrangement of the atoms. 
IV. Hematin: The iron compound united with albu- 
minous substance in oxyhemoglobin. Hemochro- 
mogen pins oxygen. 
Y. Hemin: A compound of hematin with HCI, one 
molecule of each. 
YI. Hemochromogen: Hemoglobin minus its albumin- 
ous constituent. With oxygen it gives hematin. 
A 11. Hematoporphyrin: Formed by the removal of 
iron from hematin, hemin, etc. 
hemoglobin. 
Hemoglobin, sometimes called reduced hemoglobin, 
is the coloring matter of venous blood. It contains iron, HAEMOGLOBIN. 
95 
besides the elements which enter into the composition of 
albuminous substances. The constitution of the mole- 
cule has not yet been determined, although the formulae 
of some varieties are known approximately; but it is very 
complex, containing a large number of atoms. Like the 
other members of the proteid class, it contains an albu- 
minous substance, united in this case with an iron com- 
pound. It easily unites with oxygen from the air, taking 
up one molecule of for each molecule of haemo- 
globin and forming the readily-decomposable compound, 
oxyhaemoglobin. It also forms compounds with carbon 
monoxid (CO), nitric oxid (NO), and sulphur, all of which 
are similar to its oxygen compound. 
Haemoglobin can be obtained from oxyhaemoglobin 
by the removal of the oxygen. This may be effected either 
by a vacuum, by driving it out by means of a gas which 
itself does not act on the blood, or by the use of some 
chemical reducing agent. It is obtained in the crystalline 
state with more difficulty than its oxygen compound. It 
is soluble in water, giving a reddish-purple solution. 
The spectrum of haemoglobin is of great value in test- 
ing for its presence, and the same might be said in the case 
of the haemoglobin derivatives. When a dilute solution of 
blood is held before the slit of a spectroscope, the tube being 
turned toward a window, the solar spectrum, consisting of 
the seven primary colors crossed by tine dark lines, is seen, 
and in addition one or more dark bands, which are due 
to the coloring matters of the blood. That of haemoglobin 
has one broad band with rather indistinct edges lying be- 
tween the D and E lines of the solar spectrum. If the 
liquid in the tube be shaken with air oxyhaemoglobin is 
formed, which has two dark bands. For clinical purposes 
the direct-vision or pocket spectroscope will probably be THE BLOOD. 
found to be tlic most convenient form of instrument. 
(Figs. 1 and 2 on Plate IV show the spectra.) 
A fresh alcoholic solution of guaiacum when oxidized gives 
a blue color. Many oxidizing agents, like hydrogen peroxid and 
oil of turpentine which has absorbed oxygen by standing for some 
time exposed to the air, will not act on the guaiacum alone, but 
will do so if haemoglobin or its compounds are present to serve as 
a carrier of oxygen. This is often used as a test for blood, but is 
only useful in a negative way, for protoplasm will give the same 
reaction. It can consequently be obtained from any cell, like pus 
or mucus, or even from such substances as the potato. If the 
reaction fails, however, there can be no blood present. 
Since haemoglobin is a proteid, containing an albu- 
minous substance, it will give the general reactions of 
these compounds. 
The determination of the amount of haemoglobin in 
the blood is made by comparing the color of the diluted 
blood with a solution containing a known weight of haemo- 
globin, or with some other colored liquid or solid. For 
making a standard solution of haemoglobin the recrystal- 
lized substance is used (Experiment 208). A solution of 
this can be preserved in a corked bottle or sealed tube for 
an indefinite time. The strength is ascertained by evapo- 
rating to dryness a given volume, and weighing the residue 
of haemoglobin. This method gives accurate results. 
Experiment 195. Determination of the Amount 
of Hemoglobin in Blood.—Dilute a solution containing 
a known weight of haemoglobin with distilled water until 
it is a very light-red color. Dilute in the same way the 
blood to be tested until the color is the same. Eor com- 
parison of colors the two solutions may be placed in flat- 
bottomed tubes of colorless glass (Hessler tubes) or in 
small, fiat, glass boxes, the breadth of which between the 
parallel sides is not more than a centimeter. Reckon from AMOUNT OF HAEMOGLOBIN. 
97 
the amount of dilution the amount of haemoglobin com- 
pared with the standard solution, also the absolute weight 
and percentage. 
For clinical purposes a convenient instrument for the determi- 
nation of the amount of haemoglobin in the blood is that of Fleischl, 
called an haemometer. It consists of a short, vertical cylinder for 
holding the blood, separated by a partition into two compartments; 
a long movable wedge of ruby glass under one compartment for 
a standard of color, and a white surface below for reflecting the 
light up through the wedge and cylinder to the eye of the observer. 
The amount of haemoglobin is found by filling one compartment 
of the cylinder with diluted blood, and the other, over the ruby 
prism, with water. The prism is then moved until the depth of 
color is exactly the same as that of the blood, Avhen the percentage 
of haemoglobin compared with the normal amount can be read from 
the scale. The following is the process:— 
Use for a light a lamp or yellow gas-flame, not an incandes- 
cent light or daylight. Any blood may be used for practice. In 
clinical cases use that obtained from the tip of the finger by the 
aid of a lancet. After making the incision force out a drop of 
blood by gentle pressure. Measure off the required volume of blood 
(6V2 cubic millimeters) by filling the small glass tube, open at 
both ends and mounted on a handle, which accompanies the 
hsemometer. This is accomplished by holding it horizontally and 
dipping one end into the drop. Wipe carefully all blood from the 
surface of the tube. This will be more readily done if the surface 
of the tube is kept slightly greasy by being preserved in an oily 
piece of chamois. The blood must exactly fill the tube, having 
neither a convex nor a concave surface at the open ends. 
The compartment over the ruby-glass prism is to be filled 
with distilled water by means of a pipette and the other one not 
more than a quarter full. Into the latter the open glass measuring 
tube is dipped before the blood has coagulated, and the haemoglobin 
is dissolved by moving the tube back and forth so as to Avash out 
the blood. Then rinse off the tube into the blood solution by the 
use of a few drops of Avater from the pipette. Fill the compart- 
ment from a half to three-fourths full of water and stir well with 
a Avire or Avith the handle of the measuring tube. Mix the Avater 98 
THE BLOOD. 
with the blood until no turbidity is seen and until it is evident 
that the fluid in the angles is completely incorporated with the rest. 
Now drop water from the pipette upon the blood solution until it, 
as well as the water in the other compartment, comes exactly to 
the top of the division between the compartments. If the tip of 
the pipette is placed slightly below the surface and the water flows 
slowly it will not mix with the solution of blood below. This is 
advisable in order to prevent the possibility of any of the hsemo- 
globin’s passing over into the other compartment. If both com- 
partments are filled to the top of the separating partition, so that 
there is no meniscus at the top of the liquid, they appear, when 
looked at from above, to be separated only by a narrow black line. 
A little grease on the top of the partition will help to prevent a 
mixing of the two liquids. (Some authors recommend that the 
Avater be allorved to rise above the partition and a cover glass be 
then laid on top to prevent currents.) 
Place the instrument Avith the large screw at the right, turn 
the reflector so as to illuminate the solution, shade the eye from 
other light, and move the ruby prism so that the shades of red in 
the tAvo compartments are the same. The figure in the scale, 
opposite the middle of the cylinder, gives the percentage of haemo- 
globin as compared Avith the average amount found in normal 
human blood. 
OXYIIiEMOGLOBIN. 
The crystalline form of oxyhaemoglohin differs when 
its source is from different species of animal: from human 
blood being in long prisms; from the squirrel flat, six- 
sided plates; and from the guinea-pig, tetrahedral. It 
can be easily crystallized from the blood of the dog, guinea- 
pig, and rat, hut with more ‘difficulty from human blood or 
ox-hlood. The color of the crystals is a bright red, and 
their solution is a much brighter red than that of haemo- 
globin, which, when pure, approaches a black. 
Oxyhaemoglohin is formed by the union of a molecule 
of oxygen with one of haemoglobin, and it can Avithout 
difficulty he changed hack into haemoglobin. The oxygen HAEMOGLOBIN DERIVATIVES. 
99 
is in this compound,very loosely united. Oxyhemoglobin 
may he also considered as composed of an iron compound, 
hematin, with an albuminous substance. It is decom- 
posed into these two substances when its solution is heated, 
this change being hastened acids or alkalies. When 
heated with glacial acetic acid and a little sodium chlo- 
rid it is decomposed, the hematin uniting with the nas- 
cent HCI formed at the same time and giving hemin, the 
microscopic crystals of which have a brown color and char- 
acteristic form. This is one of the best proofs for tbe 
presence of blood, although it does not distinguish between 
the different kinds. No other known substance gives 
crystals of this color and shape. 
The spectrum of oxyhemoglobin consists of two dark 
bands: a narrow one at the right of the D line in the 
yellow and a broader and less distinct one in the green 
at the left of the E line of the solar spectrum. They can 
be made to vary in width as well as distinctness by mak- 
ing the solution more or less dilute. They can be per- 
ceived when it contains 1 gramme of oxyhemoglobin in 
10 liters of water; that is, 1 part in 10,000. If to this 
solution is added a few drops of ammonium snlphid, which 
has a reducing action, the oxygen is removed and in a few 
minutes the one broad band of hemoglobin is seen in that 
part of the spectrum between the two oxyhemoglobin 
lines. It is not so distinct as are the two lines and the 
solution may have to be strengthened to make it plainly 
visible. The two lines reappear upon shaking the solu- 
tion with air, the oxyhemoglobin being formed again. 
METIIABMOGLOBTN. 
In its percentage composition methemoglobin differs 
very little, if any, from oxyhemoglobin, and probably is 100 
THE BLOOD. 
formed by a rearrangement of the atoms in the oxyhemo- 
globin molecule. It is produced whenever oxyhemoglobin 
is dried in the air at ordinary temperatures or when it is 
acted upon by weak acids. Certain oxidizing agents also 
will convert oxyhemoglobin into methemoglobin. It is 
also found in transudations and cystic fluids which con- 
tain blood; moreover in the urine during hematuria and 
hemoglobinuria, as well as in the blood itself in certain 
cases of poisoning or after a large destruction of blood- 
corpuscles by burns of the skin. 
In the methemoglobin molecule the oxygen is more 
firmly attached than in oxyhemoglobin, being removable 
neither by a vacuum nor by another gas. Like oxyhemo- 
globin, however, it is changed by weak acids or alkalies 
into hematin and an albuminous substance. Like oxy- 
hemoglobin, too, it is converted by reducing agents or by 
putrefaction, where reducing forces are at work, back into 
hemoglobin. 
Methemoglobin crystallizes in brownish-red needles 
or sometimes in plates. It is easily soluble in water, giv- 
ing a brown solution, which becomes red on the addition 
of an alkali. The spectrum of the alkaline methemoglobin 
solution has two bands much similar to those of oxyhemo- 
globin, one on the I) line, the other near the E line. 
HiEMATIN AND lIZEMIN. 
Ilematin is the iron compound which is combined 
with an albuminous substance to form oxyhemoglobin. 
It is set free whenever oxyhemoglobin is decomposed by 
the action of the gastric or pancreatic juice or by an acid. 
It is consequently found in the intestine after the eating 
of meat; also in the stomach after poisoning by a mineral 
acid. HAEMOGLOBIN DERIVATIVES. 
101 
The formula is given as:— 
C 34 or C 32 
It may he obtained from haemin, its compound with hy- 
drochloric acid. It is an amorphous substance, dark brown 
or bluish black. 
Haemin is composed of haematin and hydrochloric 
acid, probably one molecule of each. It forms micro- 
scopic crystals which, in a large amount, have a blue- 
black color. Under the microscope they are brown, rhom- 
bic prisms. They are sometimes separate, but two are often 
crossed or several are collected in clusters or rosettes. 
(Plate I, 3.) They are insoluble in water, but dissolve 
in alkalies, the haematin being set free. They are often 
called Teichmann’s crystals, and are important in proving 
the presence of blood. 
CARBONIC OX ID HAEMOGLOBIN. 
When carbonic oxid, either pure or mixed with other 
gases, is breathed or passed through blood, it unites with 
the haemoglobin, forming CO-haemoglobin: a compound 
similar to oxyhaemoglobin. It is, however, a more stable 
compound, the oxygen being unable to drive out the CO 
and take its place. Consequently the haemoglobin is ren- 
dered useless as a carrier of oxygen, and cases of poisoning 
by coal-gas, which contains carbon monoxid, are often fol- 
lowed by fatal results. The compound has a two-band 
spectrum much like that of oxyhaemoglobin, but differs in 
not being changed to haemoglobin by reducing agents. The 
crystals are similar to those of oxyhaemoglobin, but more 
of a bluish red. When mixed with strong sodium hydrate 
solution blood containing carbonic oxid gives a red mass, 
while pure blood turns brown, with a greenish cast. THE BLOOD. 
HAEMOCHROMOGEN. 
As the oxyhsemoglobin by the action of acids or alkalies is 
decomposed into an albuminous substance and haematin, so by the 
same agencies haemoglobin gives an albuminous substance and 
haemochromogen. The latter in the presence of free oxygen is 
converted into haematin. Hence, as we should expect, by the re- 
moval of oxygen from haematin by the aid of reducing agents we 
obtain haemochromogen. The spectrum of haemochromogen, in an 
alkaline solution, has two bands similar to those of oxyhaemo- 
globin, but a little farther toward the blue. The color of the 
alkaline solution is a cherry red. It is often seen in alcoholic 
specimens of the liver, muscles, etc., which have stood for a time 
in alcohol. 
lIAEMATOPORPHYRIN. 
By the action of acids upon haematin the iron is removed, 
leaving a violet to red coloring matter—haematoporphyrin. It is 
found in the contents of the stomach and intestine after poisoning 
with strong acids. It also is found in some of the dark-colored 
urines. It is insoluble in water, more soluble in acids, and easily 
so in alkalies. 
Experiment 196.—Add enough blood to a little water 
to color it a bright red. Dissolve a few crystals of ferrous 
sulphate and a little tartaric acid in a few cubic centimeters 
of water, and add a few drops to the blood solution. The 
oxyhemoglobin gives up its oxygen to the iron compound, 
becoming changed in a short time to hemoglobin, as is 
shown by the dark color. 
Experiment 197.—Shake the dark solution of hemo- 
globin with air and notice the change in color to a scarlet, 
showing the formation of oxyhemoglobin. 
Experiment 198.—Examine a very dilute solution of 
blood with the spectroscope in the following manner: 
First examine the solar spectrum by looking through the 
spectroscope with its slit directed toward a window. Close 103 
HAEMOGLOBIN DERIVATIVES. 
the slit to a very narrow opening, and focus by sliding the 
focusing tube until the fine, dark lines are seen clearly. 
There are hundreds of these so-called Fraunhofer lines in 
the spectrum of the sun, but with an ordinary instrument 
many are indistinct. A few of the most prominent should 
he noticed and used to locate the position of the dark bands 
in the spectra of haemoglobin and its derivatives. The 
most noticeable are the C line in the red, the D line in 
the yellow, the F and b not far apart in the green and F 
in the blue. If there are fine, black lines running length- 
wise of the spectrum they are caused by dust in the slit. 
Next make solutions of blood and water of different dilu- 
tions and examine the spectrum which is given when a 
test-tuheful of the solution is held before the slit after the 
solution has been shaken with air to form oxyhannoglobin. 
Notice the position of the two bands and observe that this 
does not change with the different dilutions, although the 
bands may be wider or more distinct when the solution is 
concentrated. 
Experiment 199.—To the solutions of oxyhaemo- 
globin add a few drops of ammonium sulphid, and after 
they have stood a short time examine them again with the 
spectroscope. Notice that when the oxygen has been re- 
moved Ity the ammonium sulphid the spectrum has changed 
to that of haemoglobin, which consists of one broad band 
in the space between that formerly occupied by the two. 
Experiment 200.—Shake the reduced solutions with 
air and see that the two bands have returned again. If 
any ammonium sulphid remains the oxyhannoglobin will 
he changed again to haemoglobin on standing. 
Experiment 201.—Through the solution of blood 
pass a stream of illuminating gas for a few minutes. The 
carbon monoxid is absorbed, forming carbonic oxicl Imemo- 104 
THE BLOOD. 
globin. Examine it with the spectroscope. It gives two 
dark hands much like those of oxyhemoglobin, though the 
one next the green is not as wide as in the oxyhemoglobin- 
spectrum. Try now to reduce the compound to hemo- 
globin by means of ammonium sulphid. The carbonic 
oxid is not expelled, the two bands remaining unchanged. 
Experiment 202.—Prepare an alcoholic solution of 
guaiacum by dissolving some of the gum taken from the 
middle of a lump. No blue color is produced in the solu- 
tion on the addition of a small amount of old oil of turpen- 
tine, but it is produced upon the further addition of a few 
drops of blood. 
Experiment 203.—Try the same test on the scrapings 
from a potato. A blue color is produced without the aid 
of the turpentine or any similar oxidizing agent. 
Experiment 204.—Acidify slightly a solution of 
blood, and heat to boiling. Notice the coagulated albu- 
minous substance and the dark-colored hasmatin coming 
from the decomposition of the oxyhasmoglobin. 
Experiment 205.—Filter out the hsematin or use in- 
stead a drop of fresh blood and prepare hasmin crystals 
from it by first drying thoroughly on a glass slide, then 
adding a minute amount of sodium chlorid. Cover with 
a cover glass; add a drop of glacial acetic acid, which will 
flow under the cover. Then heat over a small flame until 
the acid boils. After cooling examine under a microscope. 
The crystals sometimes are better if the acid is added and 
the slide heated two or three times. 
Experiment 206.—To prepare a large amount of hsemin, pre- 
cipitate the corpuscles from defibrinated blood by the addition of 
a large excess of a salt solution which contains 1 volume of a 
saturated salt solution in 10 to 20 volumes of water. After 
twenty-four hours pour off the solution and rinse the precipitated 105 
HAEMOGLOBIN DEB TV A TIVE S. 
corpuscles into a flask by the aid of a small amount of water. 
Add half its volume of ether, shake, and after pouring off the ether 
which removes most of the fats allow the solution of blood-coloring 
matters to evaporate in flat dishes at ordinary temperatures to a 
syrup. Mix this with 10 to 20 volumes of glacial acetic acid and 
heat in a flask one or two hours on the water-bath. Then pour 
into a beaker, add several volumes of water, and allow to stand 
several days. Wash with water and remove the albuminous sub- 
stances by boiling with acetic acid. 
Experiment 207.—-Saturate a small portion of blood 
with carbonic oxid by passing illuminating gas through it. 
Add to it twice its volume of sodium hydrate solution,—- 
specific gravity, 1.3,—containing about 27 per cent. NaOH. 
Do the same with pure blood, and spread the products on 
a piece of porcelain. Notice that the pure blood gives a 
brown color, with a shade of green. The carbon monoxid 
blood has a bright-red color on porcelain. This is a useful 
test in cases of suspected poisoning by carbon monoxid. 
Experiment 208.—Prepare crystals of oxyhsemoglobin by 
placing on a microscope-slide a drop of blood (one which crystal- 
lizes easily, like that of a dog, rat, or guinea-pig) and cover it with 
a drop of Canada balsam. Cover the whole with a cover-glass and 
examine it under the microscope. The crystals will form in a few 
minutes. They can also be made by mixing the drop of blood with 
a small drop of water on the slide and allowing it to evaporate 
until a dry ring has formed around it. Place a cover-glass over it 
and it will crystallize. It is well to examine several different 
species of blood if they can be obtained, such as guinea-pig, which 
gives crystals in the form of tetrahedra (four-sided); mouse, giv- 
ing six-sided plates; cat or dog, giving four-sided needles. 
Experiment 209.-—A large quantity of crystallized oxyhsemo- 
globin can be prepared by the following method; Make a solu- 
tion of salt containing 1 volume of saturated salt solution to 9 
volumes of water. Add 10 volumes of this to 1 of defibrinated 
blood, and let it stand a day or two in shallow, flat-bottomed 
vessels until the corpuscles have settled. Pour off the clear liquid, 
rinse the corpuscles into a separatory funnel with the aid of as THE BLOOD. 
small a quantity of water as possible, and add about as much 
ether. Shake, but not too violently, separate the solution of 
oxyhsemoglobin from the ether, and filter the former. Cool it to 
0° and mix it with one-fourth its volume of alcohol which has 
been also cooled to o°. Let the mixture stand at a temperature of 
-—2° to ■—lo° for several days, until the oxyhsemoglobin has 
crystallized. This occurs with the blood of dogs and rats almost 
immediately, but that of the ox crystallizes with much more diffi- 
culty. After crystallization filter off the crystals in the cold, and 
dry by pressing between filter-paper. The crystals may be purified 
by dissolving in a small amount of water, cooling and precipitating 
in the same manner, repeating several times. The crystals can be 
preserved for a standard in the determination of the quantity of 
oxyhsemoglobin in blood. 
Experiment 210.—To a solution of blood in water 
add a few crystals of potassium chlorate or potassium ferri- 
cyanid and warm gently. Methasmoglobin is formed, the 
color of the solution changing from a red to a brown. 
Make it slightly alkaline and it becomes red. Examine the 
spectrum of the alkaline solution. It is much like that of 
oxyhsemoglobin, though the first band is broader and ex- 
tends somewhat farther toward the red. It is reduced to 
haemoglobin by ammonium sulphid, like oxyhsemoglobin. 
Experiment 211.—Large amounts of crystalline methsemo- 
globin can be obtained by adding to a concentrated solution of 
oxyhsemoglobin enough of a concentrated solution of potassium 
ferrocyanid to give it a deep-brown color. Crystallize, as in the 
case of oxyhsemoglobin, by cooling to zero and adding one-fourth 
the volume of cold alcohol. 
Experiment 212. Preparation of ILematoporphyrin. 
—Saturate 75 grammes of glacial acetic acid with hydrobromic 
acid at 10° and add to it in a 300 cubic centimeter flask 5 grammes 
of dry hsemin crystals. Heat thirty minutes on a water-bath 
until no more hydrobromic acid fumes escape, then pour into a 
liter of water. After standing several hours filter and to the red 
filtrate add sodium hydrate till the liquid is neutral. The coloring 
matter is precipitated and should be filtered off, washed, and 107 
TESTING OF STAINS. 
drained upon filter-paper. Digest the moist precipitate with dilute 
sodium hydrate on the water-bath; filter off the hydrate of iron, 
which separates, and allow the solution to stand until the sodium 
compound of hsematoporphyrin has separated in crystalline 
masses. Dissolve these in water and precipitate the coloring 
matter by acidifying with acetic acid. Filter, wash with water, 
and after stirring the precipitate up to a paste with a small 
quantity of water dissolve by adding hydrochloric acid carefully. 
Evaporate the dark-red solution in a vacuum over sulphuric acid. 
The hydrochloric acid compound of hsematoporphyrin crystallizes 
in brownish-red needles. 
Experiment 213.—Examine the spectrum of the hsemato- 
porphyrin. 
Experiment 214.—Dissolve a few hsemin crystals in water 
by the aid of sodium hydrate and examine the spectrum. 
Experiment 215.—Convert the hsemin into hsemochromogen 
by removing the oxygen. Use for this purpose a solution of fer- 
rous sulphate with tartaric acid, the whole being made alkaline 
with ammonia. Examine the spectrum. 
Experiment 216.—If hsemin crystals are not at hand, pre- 
pare the hsemochromogen from a dilute solution of blood by first 
making it alkaline with sodium hydrate, then reducing the hsema- 
tin thus formed by ammonium sulphid or ammoniacal ferrous tar- 
trate. 
Testing Suspected Stains foe Blood. 
The following tests can he used, first on a stain made 
by drying a drop of blood on a piece of cloth, then upon 
unknown stains. 
1. Soak a small portion of the stain in a few drops 
of water on a microscopic slide. If no color is imparted 
to the liquid, blood is not present or the haemoglobin has 
been so much decomposed that only the haemin test will 
show its presence. 
3. If a red color was seen in the water try the spec- 
troscopic test. By drying, both haemoglobin and oxy- 108 
THE BILE. 
haemoglobin are changed into methasmoglobin, which 
gives a somewhat different spectrum. (Plate IV, 12.) 
Ammonium sulphid reduces this to haemoglobin and 
shaking with air gives the spectrum of oxyhaemoglobin 
(Experiments 198 and 199). If these are all obtained the 
presence of blood is proved. 
3. To confirm the results, or if the stain is too small 
to obtain them, try to obtain the haemin crystals as in 
Experiment 204. After applying the acid to the dried 
mass the latter should be broken up with a glass rod to 
insure thorough mixture. These haemin crystals are pro- 
duced only from the coloring matters of the blood. When 
in doubt as to their identity they should be compared with 
those obtained from known blood, remembering that dif- 
ferent specimens of crystals may differ considerably in 
size. 
4. If there is a sufficient amount of the blood, not 
too long exposed to the air, or if it is desirable to de- 
termine the species of animal, it may be soaked in a small 
amount of Wper-eent.-salt solution and -the appearance 
and size of the corpuscles compared with those of known 
specimens or measured by a microscope with a micrometer 
eye-piece. 
THE BILE. 
The bile is normally a brown to greenish, viscid fluid 
with a bitter taste and a neutral or slightly alkaline re- 
action. It is a mixture of the secretions of the liver-cells 
with that from the mucous membrane of the passages, 
which latter contains a viscous substance .similar to the 
nhcleoalbnmins. This is sometimes called biliary mucin, 
although it differs in some respects from true mucin. 109 
THE BILIAEY ACIDS. 
The compounds which make up the larger part of 
the solid matters of the bile are the sodium salts of gly- 
cocholic and taurocholic acids. Besides these and the 
biliary mucin there are present fats, soaps, lecithin, and 
cholesterin, also a number of inorganic salts of the alka- 
lies, alkaline earths, and iron. The color of the bile is 
due to the biliary pigments, bilirubin, biliverdin, etc. 
The salts of the biliary acids in the Bile of different 
animals vary in their proportions. In the case of car- 
nivorous animals only the taurocholic acid is found; in 
the human bile, as well as that of most cattle, both are 
present. The biliary acids are both compounds of cholic 
acid, C 24 Glycocholic acid, C 26 is com- 
posed of cholic acid united with glycocol, CH2NH2C02H; 
taurocholic acid, C 26 of cholic acid and taurin, 
C2II4NH2S03H. They can be decomposed into their con- 
stituents by the caustic alkalies. With cane-sugar and 
sulphuric acid the biliary acids give a purple color, and 
this can be used as a test of their presence. 
This test is an extremely-delicate one, and its failure indi- 
cates that biliary acids are absent. There are, however, other 
substances—like albumin, morphine, and amyl-alcohol—which give 
a similar color. In these cases the spectroscopic test should not be 
neglected. The biliary acids can be obtained pure by evaporating 
the solution to dryness, extracting with absolute alcohol, pre- 
cipitating this solution with ether, and applying the test to the 
precipitate. The purple solution, when sufficiently diluted with 
alcohol and examined spectroscopically, gives a dark band between 
D and E, near to E, and another before F. These are not seen 
with albumin, etc. 
11l concentrated sulphuric acid they give a green color, 
showing a strong fluorescence. The sodium salts are ob- 
tainable from the bile by evaporating to dryness and, after 
dissolving in alcohol, precipitating with ether. 110 
THE BILE. 
Cholesterin, C 26 occurs in most of the fluids 
of the body, as well as in the hile, and the calculi or 
concretions of the gall-bladder, of which it forms the 
principal part. It is not common in the urine, but is a 
constant ingredient of the faaces. it is insoluble in water, 
hut soluble in ether, chloroform, or hot alcohol. It crys- 
tallizes from ether in fine, silky needles; from alcohol in 
large plates containing a molecule of water of crystalliza- 
tion. (Plate I, 4.) In large quantities it has the appear- 
ance of a mass of white plates with a pearly luster and a 
greasy feeling. It is distinguished from the fats by its 
insolubility in the caustic alkalies, even when boiling. It 
forms compounds with the fatty acids similar to the fats, 
the cholesterin taking the place of the glycerin. Lanolin, 
which is found in wool-fat, is an example. These are not 
easily decomposed by bacteria, hence can he advanta- 
geously substituted for the animal fats where decomposition 
is objectionable. The cholesterin as found in the animal 
body seems rather to be an excrementitious material than 
to have any function of its own. 
The bile contains at least two well-characterized pig- 
ments or coloring .matters: bilirubin, C 32 and 
biliverdin, C 321 The different colors of hile from 
a brown to a green are due to a preponderance of one or 
other of these. They seem to be formed from the blood- 
coloring matters, being found in old blood-extravasations 
and being increased in amount in the bile when the blood- 
corpuscles are destroyed, so that the coloring matters are 
set free in the plasma. 
Bilirubin occurs in many biliary calculi, particularly 
in and around the nucleus. This is the best source of the 
pure substance. It is commonly an amorphous powder, 
orange red in color. It is insoluble in water, but can he THE BILIARY PIGMENTS. 
dissolved in chloroform, and crystallizes from the latter in 
plates and prisms. It unites with strong bases and in cal- 
culi occurs in union with calcium. By reduction hydro- 
bilirubin, CB3H40N4O7, is formed. This change takes place 
in the large intestine as a result of putrefactive action, and 
the color of the faeces is due principally to the hydrobili- 
iTihin. Bilirubin is acted upon by oxidizing agents, with 
the formation of biliverdin. This change takes place when 
an alkaline solution is left exposed to the air. 
Biliverdin is an amorphous, green powder. It differs 
from bilirubin in being insoluble in chloroform, and the 
two can consequently he separated by this reagent. 
Both of these biliary pigments when acted upon by 
yellow nitric acid, such as is formed by allowing the strong 
acid to stand in a bright light, undergo a change of color 
through green, blue, violet, and red to yellow. 
Besides these two coloring matters a number of others 
have been described by different authors. Of them com- 
paratively little is known. They appear to be derived from 
biliverdin and bilirubin, and it is to their formation that 
the play of colors is due when bile is acted upon by oxid- 
izing agents. Some of these are:— 
Biliprasin, greenish black. 
Bilifuscin, brown. 
Bilicyanin, blue. 
Choletelin, yellow to brown. 
Not infrequently there are found in the gall-bladder con- 
cretions, commonly known as gall-stones. They are sometimes 
nearly as large as a hen’s egg, and may fill the bladder almost com- 
pletely. They are soft and often worn away from rubbing against 
one another. If they are cut through the nucleus is generally 
found to be dark colored and composed of bilirubin-calcium. 
Around this are concentric layers, usually of cholesterin, but some- 
times of the bilirubin-calcium. Calcium carbonate is also found in the concretions, as well as others of the biliary pigments in smaller 
amounts. 
THE BILE. 
Experiment SI7. Separation of the Salts of 
the Biliary Acids.—The contents of the gall-bladder 
of an ox should be mixed with washed sand and evapo- 
rated to dryness on the water-bath. Then pulverize the 
mass in a mortar, which operation is facilitated by the 
sand. Dissolve the biliary salts by strong alcohol, and 
filter. Evaporate this solution to a small volume on the 
water-bath, allow it to cool, pour it into a flask, and pre- 
cipitate with an excess of ether, shaking, to mix thor- 
oughly. After standing a. few hours the precipitated mass 
is converted into clusters of silky crystals (sometimes called 
crystallized bile). These are a mixture of the sodium salts 
of the tanrocholic and glycocholic acids. They can be 
purified by filtering, washing with water, dissolving in the 
smallest possible quantity of water, and precipitating again 
with ether. They crystallize then in long, thin, colorless 
crystals with a silky luster. 
Experiment 318. Pettenkofer’s Test for the 
Biliary Acids.—-Use the salts or the ox-bile. Mix in a 
porcelain dish or test-tube with a small amount of con- 
centrated sulphuric acid, being careful not to let the tem- 
perature rise above 70° C. It must, however, be above 
50°. Then add, drop by drop, a 10-per-cent, solution of 
cane-sugar, stirring with a glass rod. A red color appears. 
If too much sugar is added or the temperature is too high 
the sugar is decomposed by the acid, giving dark-brown 
products, which conceal the red color. 
Experiment 319.—The same result may be obtained 
by adding the sugar to the liquid to be tested, acidifying 
with dilute sulphuric acid, and dipping into it a piece of 
filter-paper. Allow the paper to dry, or dry it at a mod- THE BILIARY ACIDS. 
113 
erate heat, to avoid charring. When it is completely dry, 
the red color appears on the paper. If heated too highly 
it will be turned black by the acid. 
Experiment 220.—Examine the spectrum of the colored 
liquid obtained in 218. There are two absorption-bands: one at F, 
the other between D and E, near E. 
Experiment 221. Preparation of the Free Biliary 
Acids.—Dissolve in water some of the sodium salts obtained in 
217. Add dilute sulphuric acid to it slowly, until a precipitate 
commences to form, then add a little ether. After standing in the 
cold until the acid has separated, filter, wash, and recrystallize 
from a small amount of hot water. This gives the glycocholic 
acid. 
Experiment 222. Preparation op Cholic Acid.—Add 
to ox-bile one-fifth of its weight of 30-per-cent, sodium hydrate 
and boil for twenty-four hours, adding more water to replace that 
which has evaporated. Then saturate the liquid with carbon di- 
oxid, evaporate to dryness, and extract the mass with strong 
alcohol. The sodium salt of cholic acid dissolves, as well as some 
other sodium compounds. Dilute with water until the solution 
contains no more than 20 per cent, of alcohol, then precipitate 
with dilute barium ehlorid as long as there is a precipitate. Filter 
and test the filtrate with barium ehlorid, which must give no pre- 
cipitate. Then precipitate the cholic acid from tnis filtrate by de- 
composing its sodium salt by means of hydrochloric acid. Let it 
stand several hours until it has become crystalline, then recrystal- 
lize from alcohol. 
Experiment 223.—Test the cholic acid thus obtained with 
concentrated sulphuric acid, and notice that it gives a green fluo- 
rescence. Add a few drops of a cane-sugar solution and see that a 
red color appears, as with the undecomposed biliary acids. 
Experiment 224. Preparation of Taerocholic Acid.— 
For this purpose use dogs’ bile, which contains the sodium salt 
of the acid. Evaporate, dissolve in alcohol, and precipitate with 
ether as in Experiment 217. The acid can be set free by adding 
dilute HLSO4, but is very soluble in water and difficult to' obtain 
in the crystallized condition. 
Experiment 225.—Add the solution of the taurocholic acid 114 
THE BILE. 
Acidified with IbSO, to solutions of albumin or peptones and ob- 
serve that they form insoluble compounds. 
Experiment 226. Preparation of Taurin.—Mix ox-bile 
with an excess of concentrated hydrochloric acid, and evaporate 
the liquid to a small volume by boiling. Pour off the solution 
from the resinous mass of acids which have separated, and evapo- 
rate this liquid until the sodium chlorid has, for the most part, 
crystallized out. Filter, and mix the filtrate, after cooling, with 
strong alcohol. The taurin is precipitated. Filter and wash out 
the salt from this with a little water. Dissolve the precipitate in 
the smallest possible quantity of hot water. On cooling the taurin 
crystallizes out in four-sided prisms. 
Experiment 227. Preparation oe Cholesterin.— 
Biliary calculi or gall-stones are the best source of cho- 
lesterin. Powder the calculus, remove the hile by boiling 
water, then dissolve the cholesterin in boiling alcohol, and 
filter while hot. It separates from the filtrate on cooling. 
The insoluble residue, which consists largely of compounds 
of the biliary coloring matters, can be used for the prepara- 
tion of these. The cholesterin may be further purified by 
dissolving it in an alcoholic solution of potassium hydrate 
with the aid of heat. After it separates on cooling, wash 
well with water on the filter, then recrystallize from a 
mixture of alcohol and ether. 
Experiment 228.—Examine the crystals under the 
microscope. They are in the form of large rhombic tables 
or plates. 
Experiment 229.—T0. a crystal of cholesterin in a 
test-tube or under the microscope add a drop of concen- 
trated sulphuric acid, then a drop of iodin solution. The 
crystal becomes first violet, then blue, green, and red. 
Experiment 230.—Dissolve a crystal of cholesterin 
in a few drops of chloroform in a test-tube, then add an 
equal volume of concentrated sulphuric acid. The chloro- O.HOLESTERIN. BILIARY PIGMENTS. 
115 
form solution becomes red, then cherry red, and purple. 
On pouring it into a dish it becomes blue, green, and 
finally yellow. 
Experiment 231.—Evaporate on a piece of porcelain 
a small crystal of cholesterin with a drop of concentrated 
nitric acid. A yellow stain remains which, if treated while 
warm with ammonia, gives a red color. Too high heating 
prevents the reaction. 
All these reactions can be employed for the identifica- 
tion of cholesterin. 
Experiment 232. Preparation of the Biliary 
Pigments.—lf biliary calculi are not available, bile may 
be used for obtaining bilirubin, employing the yellow or 
brown in preference to the green. Dilute it with a little 
water and add a small amount of lime-water, avoiding an 
excess. Mix by shaking. Pass through the liquid a stream 
of carbon dioxid to convert any excess into calcium car- 
bonate. Filter out the bilirubin, which has been precipi- 
tated as the calcium compound, and wash it with water. 
Suspend the precipitate in water, decompose it with a slight 
excess of hydrochloric acid, and shake it immediately with 
a small amount of chloroform to take up the free bilirubin, 
otherwise it will oxidize to biliverdin. Separate the chlo- 
roform solution from the water and precipitate the bili- 
rubin from it by alcohol. 
Experiment 233.—1f biliary calculi are at hand they 
may be used instead of the bile. Pulverize the calculi, then 
dissolve the cholesterin with a mixture of alcohol and ether. 
(The residue from the alcoholic extraction in the prepara- 
tion of cholesterin may be used [Experiment 227].) After 
the cholesterin has been removed decompose with acid and 
proceed as in the preceding experiment, 232. 
Experiment 234.—Convert a portion of the bilirubin 116 
THE BILE. 
into biliverdin by dissolving in dilute sodium hydrate and 
letting the solution stand in an evaporating dish. When 
it has turned green, precipitate with an excess of hydro- 
chloric acid, filter, and wash. 
Experiment 235.—The biliverdin in an impure state can be 
obtained from ox-bile by precipitating the mucin with several times 
its volume of alcohol, then precipitating the biliverdin by barium 
chlorid. Filter, wash with water, and alcohol, then decompose 
with hydrochloric acid. The biliverdin is insoluble in the acid. 
To remove the fat it must be extracted with ether, then the bili- 
verdin can be dissolved in alcohol, which, after filtering, is left to 
evaporate. 
Experiment 236. Gmelin’s Test.—To a solution 
of bilirubin in dilute alkali add slightly yellow, concen- 
trated nitric acid, holding the tube in a slanting position 
and pouring slowly so that the acid flows down under the 
bilirubin solution. Notice the colored rings: green nearest 
the top, then blue, violet, red, and yellow next to the acid. 
The acid must not be too yellow or the pigments quickly 
oxidize and nothing is seen but a yellow color. 
Experiment 237. Huppert’s Test.—To an alkaline 
solution of bilirubin add lime-water to precipitate the bili- 
rubin. Filter, wash with water, place in a test-tube half 
full of alcohol slightly acidified with sulphuric acid, and 
boil for some time. The bilirubin is oxidized to biliverdin 
and the alcohol becomes colored green or bluish green. 
Experiment 237 a.—To a little of the bilirubin solu- 
tion in a test-tube add a dilute tincture of iodin so that 
it floats on top. An emerald-green ring is seen between 
the liquids. 
BONE. 
Bone contains an organic compound, collagen, and a 
number of inorganic or mineral substances. These latter BONE 
117 
are the phosphates of calcium and magnesium, mainly the 
former; also calcium carbonate and small amounts of cal- 
cium ehlorid and fluoric!. The inorganic substances can 
be removed by acids, leaving the hone flexible. If a bone 
is heated the collagen is decomposed, with an evolution of 
ammonia, showing that the collagen is a nitrogenous com- 
pound. Then inflammable gases are set free. If the igni- 
tion is performed where free access of air is prevented, 
there remains a black mass known as bone-black or animal 
charcoal. The black color is due to carbon, which can be 
removed by burning in the air, leaving the mineral or in- 
organic constituents only. The bone-black is an extremely 
porous substance and has the power of absorbing from 
their solutions many of the vegetable coloring matters and 
also the alkaloids. On this account it is used for decolor- 
izing liquids as well as for an antidote in cases of poison- 
ing by strychnine and some other alkaloids. 
Experiment 238.—Fill a dry test-tube one-third full 
of fragments of dry bone, fasten it horizontally by the 
upper end in a clamp, and heat, at first gently, then to as 
high a temperature as possible without softening the glass, 
moving the burner so as not to heat it in one spot. The 
organic matter is decomposed. First water is given off, 
then ammonia. Test this with a piece of red litmus- 
paper. An oily or tarry mixture distills off with inflam- 
mable gases. When the gas has been expelled the mineral 
matters of the bone remain mixed with carbon. 
Experiment 239.—Take a piece of this and heat it 
in the air, holding it with the forceps or a piece of wire. 
The carbon burns away, leaving only the mineral matters 
as a brittle mass. 
Experiment 240.—Dissolve the bone-ash in dilute BONE 
nitric acid. Notice that carbonates are present, as shown 
hy the effervescence of carbon dioxid gas. 
Experiment 241.-—Test a portion of the solution for 
phosphoric acid, which is present in the phosphates, by 
making it strongly acid with nitric acid, then adding am- 
monium molybdate and warming gently. A yellow pre- 
cipitate shows phosphoric acid. 
Experiment 242.—-Test another small portion of the 
solution for chlorids with silver nitrate. They give a milk- 
iness or a white precipitate, but are present in very small 
quantities. 
Experiment 243.—Test the remainder of the nitric 
acid solution for calcium and magnesium after removing 
the phosphoric acid in the following manner: Add a few 
drops of ferric chlorid to the solution in a beaker. The 
iron unites with the phosphoric acid, which was held by 
the calcium and forms ferric phosphate. Four a few drops 
from the beaker into a test-tube and test hy adding am- 
monia to see if all the phosphoric acid has united with the 
iron. If this is the case, the ammonia gives a yellowish 
precipitate. If not enough ferric chlorid was added the 
precipitate is white, and more of the iron solution must 
be added and the test repeated until all the phosphoric 
acid has been taken hy the iron. Then to the solution add 
a solution of sodium carbonate until it is nearly neutral; 
that is, until the precipitate—which forms as the sodium 
carbonate strikes the liquid—dissolves slowly on stirring. 
Then add 1 or 2 grammes of barium carbonate to pre- 
cipitate the ferric phosphate. Filter after warming. Pre- 
cipitate the barium from the hot filtrate with dilute sul- 
phuric acid and filter. Having thus removed the phos- 
phoric acid, test the filtrate for calcium hy making it alka- 
line with ammonia and adding ammonium oxalate as long 119 
MUSCULAR TISSUE. 
as a precipitate is formed. The calcium is thrown down 
as white calcium oxalate. Filter and to the filtrate add 
sodium phosphate. A white crystalline precipitate shows 
magnesium. 
Experiment 244.—Test the absorptive power of bone-black 
by adding it to a light-blue solution of indigo and wanning, then 
filtering. The coloring matter will have almost or entirely disap- 
peared from the filtrate. 
Directions have been given before (Experiment 113 a) 
for the separation of the collagen from the mineral con- 
stituents of the bone. 
MUSCULAR TISSUE. 
A muscle is made up of fibers or cells, consisting of 
a sheath (the sarcolemma), composed of a substance similar 
to elastin, and its contents. The latter are mostly albu- 
minous matters, alkaline and liquid during life, but be- 
coming acid and more solid after death. This albuminous 
liquid, which in many respects corresponds to the plasma 
of the blood, is called muscle-plasma. It coagulates quickly 
at the ordinary temperatures, and thus gives rise to the 
rigor mortis of the muscles after death. The coagulated 
mass is myosin; a mixture of a number of compounds. 
The cause of the coagulation of the myosin in this case 
is probably the formation of lactic acid, which accumulates 
in the muscle after death. 
If fresh muscular tissue is treated with boiling water, 
most of the albuminous substances are coagulated and 
upon filtering remain with the fats in the insoluble residue. 
The solution contains, besides inorganic matters, a class 
of organic compounds, often called, from the method of 
obtaining them, the “extractives.” They may be divided 120 
MUSCULAR TISSUE. 
into two groups: those which contain no nitrogen and 
those of which nitrogen is a constituent. Among the non- 
nitrogenons are lactic acid and its compounds; also gly- 
cogen and its derivatives: dextrin, maltose, and glucose. 
The principal ones of the nitrogenous extractives are 
creatin and creatinin, small quantities of urea and uric 
acid, and the nuclein bases, such as gnanin, xanthin, and 
hypoxanthin—formerly called sarcin. Carnin, which is 
similar in properties and composition to the nuclein bases, 
is also found in the watery extract of muscle. 
Creatin 
NHo 
NIT = C 
N OIL. CH2 COJI 
NH CO 
NH = C 
nch3 ch2 
and creatinin: 
are closely related, the latter being derived from the former 
l>3r taking away one molecule of water, and can be changed 
hack into creatin by adding the water again. 
The nuclein bases, gnanin (CSH5jSTSO), hypoxanthin 
(CSH4N4O), and xanthin (C5H4N402) are similar in their 
properties and related to uric acid (CSH4jSr4O3). They 
occur partly free in the muscular tissue and partly united 
with phosphoric acid and albuminous substances in the 
form of nucleins. All these soluble compounds are found. MUSCLE-PLASMA. 
121 
naturally, in the various meat-extracts, which are used in 
foods. They are probably formed in the body by the de- 
composition of the albuminous materials, but their office 
in the vital processes is not clearly understood. When 
taken as a food, their value is rather in the stimulation of 
digestion through their agreeable taste than in their abso- 
lute nutritive worth. This may be merely because of their 
increasing the secretion of the digestive fluids. 
Experiment 245. Preparation op Muscle-plasma.—Kill 
a frog and immediately wash the blood from the body by passing 
in through a cannula inserted in the aorta a cold 0.5-per-cent, 
sodium ehlorid solution. The necessary force can be gained by 
placing the solution in a doubly-tubulated bottle, which can be 
raised and lowered, and connecting the lower tubulure with the 
cannula by a small rubber tube. Cut the muscle up as quickly 
as possible with a cold knife or pair of scissors and freeze it by 
stirring in a beaker, previously surrounded by a freezing mixture 
of ice (3 parts) and salt (1 paid). It freezes at about —7° C. 
Then rub it to as fine a powder as possible in a mortar, which, 
as well as the pestle, has been cooled below this temperature by 
standing in a freezing mixture. Subject the mass to a strong press- 
ure, which gives a yellowish liquid. Filter this through muslin 
at a temperature below freezing. The filtrate is the muscle-plasma. 
Through the whole process care must be taken to preserve a low 
temperature to prevent coagulation. 
Experiment 246.—Pour a few drops of the plasma into a 
dish of the ordinary temperature. It coagulates immediately. 
Experiment 247.—Test the reaction of the plasma to litmus- 
paper. It is alkaline. 
Experiment 248.—Allow the temperature of the rest of the 
plasma to rise slowly, and notice that it coagulates at a little above 
freezing. On standing a yellowish liquid is pressed out of the clot, 
as in the ease of the coagulum of blood-plasma. This is muscle- 
serum. 
Experiment 249.—Try the reaction of the muscle-serum to 
litmus-paper. It is alkaline. MUSCULAR TISSUE 
Experiment 250.—Prove that the coagulated mass is a 
globulin (Experiments 77, 78, and 79). 
Experiment 251.—Take about 500 grammes of lean beef, 
and, after removing, as completely as possible, the fat and con- 
nective tissue, chop it finely. Add an equal weight of water and 
heat half an hour on a water-bath to 55° or 60°. Filter through 
muslin, pressing out the water with the hands. Repeat the ex- 
traction with half as much water. Unite the filtrates, and boil to 
precipitate the albuminous compounds. Filter and add lead ace- 
tate as long as a precipitate forms, avoiding a great excess. Filter 
and remove the lead by passing hydrogen sulphid gas into the solu- 
tion. Filter out the lead sulphid and evaporate the filtrate on the 
water-bath to sor 10 cubic centimeters. Allow the yellowish syrupy 
liquid to stand two or three days in a cool place, when the creatin 
crystals will separate. Filter, and wash with 88-per-cent, alcohol. 
Unite the filtrate and the washings and remove the alcohol by 
evaporation on a water-bath. After cooling make alkaline with 
ammonia and add an ammoniacal solution of silver chlorid. The 
precipitate contains the silver compounds of hypoxanthin, xanthin, 
and yuanin. (The filtrate contains lactic acid. Preserve for test- 
ing.) Filter. Wash with ammonia and dissolve in boiling HNOa, 
sp. gr., 1.1, to which a little pure urea has been added to prevent 
the decomposition of the bases. While hot, filter from a small 
amount of AgCl, which may remain, then allow to stand twelve 
hours. Hypoxanth,in-silver nitrate separates in small needle-shaped 
crystals. Filter and wash with water. From the filtrate, by the 
addition of an excess of ammonia, is obtained a slight precipitate of 
xanthin-silver oxid. The free xanthin and hypoxanthin may be ob- 
tained by suspending their silver compounds in water and, after 
heating and making slightly alkaline with ammonia, adding am- 
monium sulphid drop by drop until the silver is precipitated, avoid- 
ing an excess. On evaporating the filtrate the xanthin and hypo- 
xanthin will be left as microscopic crystals. 
Most of the guanin is left in the precipitate made by the am- 
monium sulphid. It can be dissolved by boiling with a little very 
dilute hydrochloric acid. Filter and precipitate it from the filtrate 
by making it alkaline with ammonia. 
To obtain the lactic acid from the filtrate from the precipitated 
hypoxanthin, etc., first precipitate the silver by FLS and filter. 123 
THE EXTRACTIVES. 
Concentrate the filtrate on the water-bath until most of the am- 
monia has been expelled. Then cool and acidify strongly with 
dilute sulphuric acid. The lactic acid is thus set free and can now 
be separated by shaking gently with about one-fifth its volume 
of ether, which dissolves the lactic acid, but not the sulphuric. 
After shaking in a glass-stoppered funnel, allow it to stand until 
the ether has all risen to the top of the liquid. Then draw off 
the water and the ether into separate flasks. Repeat the operation 
a few times with fresh portions of ether. Mix the different por- 
tions of ether and distill or evaporate it. The residue contains 
the lactic acid mixed with a little sulphuric. Dilute with water 
and boil a minute with zinc carbonate until it has lost its acid 
reaction. Filter, and evaporate the filtrate on the water-bath to a 
small volume. Then let it stand, and the zinc lactate will crystal- 
lize in four-sided prisms: (C3HSO3)2Zn + 2ELO. Filter these from 
the remaining liquid, and dry on filter-paper. 
To obtain the free acid dissolve some of the crystals in water 
and precipitate the zinc Avith hydrogen sulphid gas. Filter, and 
evaporate the filtrate. The acid will be left as a syrupy liquid. 
Test it for its acid reaction and sour taste. It differs from fer- 
mentation lactic acid in that it rotates the plane of polarized light 
toward the right. Fermentation lactic acid does not do this. 
Experiment 252.—Convert a part of the creatin into cre- 
atinin by boiling fifteen minutes Avith very dilute sulphuric acid. 
Neutralize the acid by adding poAvdered barium carbonate as long 
as it effervesces. Evaporate to dryness on a Avater-bath and ex- 
tract the creatinin from the residue with strong alcohol. Upon 
evaporating, the creatinin is left in the form of crystals. 
Experiment 253.—Dissolve a little of the creatinin in a small 
amount of Avater, add a solution of zinc chlorid, and alloAV to stand. 
Characteristic crystals in clusters or rosettes appear. They are a 
double salt of creatinin and zinc chlorid. 
Experiment 254.—T0 a creatinin solution add a few drops of 
a freshly-prepared solution of sodium nitroprussid, then, drop by 
drop, dilute sodium hydrate. The liquid becomes ruby red, soon 
changing to straw color. If it is now strongly acidified with 
acetic acid and boiled, it becomes green, then blue. 
Experiment 255.—To a solution of creatinin add a few drops 124 
MILK 
of sodium hydrate, then of picric-acid solution. A red color is 
obtained. 
MILK. 
The solids of milk are partly dissolved and partly in 
suspension in the liquid. Of the dissolved constituents the 
most important are milk-sugar, an albumin, a globulin, and 
some mineral salts. Among the suspended compounds 
are casein, fat, and calcium phosphate. The average 
amount of solids in normal cows’ or human milk is 12 or 
13 per cent, by weight. The reaction of fresh cows’ or 
human milk is nearly neutral, or may be anrphotere to 
litmus; that is, it turns red paper blue and blue paper red. 
The specific gravity should be between 1.029 and 
1.033 at 15°, and of milk which has been skimmed after 
standing twenty-four hours it should be between 1.0325 
and 1.0365. Thus the removal of fat raises the specific 
gravity and the addition of water lowers it. The average 
percentage composition of milk is given by Konig as fol- 
lows : 
Cows’,—water, 87.17; proteins, 3.55; fats, 3.69; lac- 
tose, 4.88; mineral matter, 0.71. 
Human,—water, 87.41; proteins, 2.29; fats, 3.78; lac- 
tose, 6.21; mineral matter, 0.31. 
Casein, which is a nucleoalbumin, is not in true solu- 
tion in milk, since it can be filtered out by unglazed por- 
celain, though not by filter-paper. It is precipitated by 
weak acids, as is seen when the milk becomes sour, but is 
not coagulated by boiling. Eennin breaks up the casein 
into two compounds: an albmnose and an insoluble cal- 
cium compound (paracasein calcium, or cheese). Coagu- 
lated human casein is not as hard as that of cows. The 
difference is partly due to its chemical composition, but MILK 
largely to the fact that cows’ milk contains more casein 
and calcium than human milk. It can he made to form 
a soft and spongy coagulum similar to the human hy dilu- 
tion or hy the removal of the calcium compounds. 
The fats of milk are a mixture of stearin and olein, 
with a small amount of the glycerids of some lower mem- 
bers of the fatty-acid series,—butyric, caproic, caprylic, 
capric, etc. The fat exists as an emulsion, a coating of 
albumin keeping the globules separate. They may he made 
to collect hy dissolving this coating hy a chemical agent, 
like sulphuric acid. Babcock’s method for determining 
the percentage of fat in milk is based upon this principle. 
Here the volume of the fat is measured and this gives the 
relative amount in the milk. If the fat rises for twenty- 
four hours without such decomposition it should form a 
layer 10 or 15 per cent, of the depth of the milk, if the 
latter is normal. 
The method of obtaining the milk-sugar has been 
given. (Experiment 33.) 
Experiment 256.—Test the reaction of fresh milk to 
red and blue litmus-paper. 
Experiment 357. Determine the specific gravity 
with an accurate urinometer. 
Experiment 258.—Remove the fat from milk by a 
centrifuge, or after standing, and determine the specific 
gravity again. 
Experiment 259.—Try the same test after adding 
from 10 to 35 per cent, of water to the milk. 
Experiment 260.—In a weighed porcelain or platinum cru- 
cible evaporate 10 cubic centimeters of milk to dryness on a water- 
bath and weigh quickly to find the amount of the total solid 
matter. The drying will take place much more rapidly if a 126 
MILK. 
weighed quantity (about 20 grammes) of dried sand is added, but 
the residue cannot be used for the next experiment. 
Experiment 261.—Heat the dried substance in the crucible, 
at first gently, then until no black remains. The residue is the 
mineral matter, or ash. There should not be over 1 per cent, of the 
weight of the milk. 
Experiment 263.—Compare tire action of remain 
upon cows’ and human milk. Try the remain in cows’ 
milk to which 50 per cent, of water aiad a few drops of 
ammonium oxalate have been added to remove the calcium 
salts. 
Experiment 263.—To separate the nitrogenous con- 
stituents of milk first precipitate the casein by saturating 
the milk with sodium chlorid. Filter and to the filtrate 
add powdered magnesium sulphate as long as it dissolves, 
stirring meanwhile. This precipitates paraglobulin: the 
same compound that is found in the blood. Filter and 
apply the globulin tests (Experiments 78 and 79). Acidify 
the filtrate Avith a few drops of dilute acetic acid and boil. 
The albumin of milk—lactalbnmin—is coagulated. 
Experiment 264.—Examine a drop of milk under a 
cover-glass with the microscope. 
Experiment 265.—Destroy the emulsion by adding 
10 cubic centimeters of concentrated sulphuric acid to an 
equal volume of milk. Let it stand, and the fat rises to 
the top in large globules. The separation is complete in 
a feiv minutes if a centrifuge is used. The volume of the 
fat can be better seen by using a narrow-necked flask and, 
after mixing with the acid, nearly filling with warm water. 
Experiment 266.—Fill a 100-cnbic-centimeter grad- 
uated cylinder to the upper mark with milk and let it 
stand hours. There should be 10 or 15 cubic 
centimeters of cream. THE URINE. 
Experiment 367. To Determine the Percentage 
op Lactose in Milk.—Dilute 30 cubic centimeters of 
milk to 400 cubic centimeters. Drop in acetic acid slowly 
until it coagulates; then pass carbon dioxid gas into the 
liquid fifteen minutes and let it stand until it settles clear. 
Filter and wash; coagulate the albumin and globulin in 
the filtrate by boiling. Filter, wash, and use the filtrate or 
a part of it for the sugar-determination by Fehling’s solu- 
tion as in the determination of glucose (Experiment 33). 
For every 10 cubic centimeters of the solution which is 
decolorized 0.067 gramme of lactose is present. 
THE URINE. 
The urine is a solution which contains the final prod- 
ucts from the chemical changes in progress in the animal 
body. A part of these are excreted in the expired air and 
from the skin, and a still smaller part through the mucous 
membrane of the intestine, but, if we omit the carbon di- 
oxid from the lungs, by far the greater proportion of these 
final products is found in the urine. A study of its com- 
position and variation, therefore, is often of great value 
in judging of changes which are going on in the body. 
Among the most common inorganic constituents 
normally found are the chlorids, sulphates, and phosphates 
of sodium, potassium, calcium, and magnesium. Of the 
normal organic compounds there are urea, uric acid and 
its salts, creatinin, etc. The following, when found in 
more than minute amounts, may be regarded as patho- 
logical: Glucose, albuminous substances, blood, bile, pus, 
fat, mucin, leucin, and tyrosin. Others which are more 
rare will be spoken of later. All of these either are taken 128 
THE URINE. 
as such into the body with the food or are formed in the 
body by chemical action. The significance of each may 
depend upon the amount which is present, as well as upon 
its mere presence or absence. In interpreting the mean- 
ing of each of the constituents of the urine its method of 
formation must be considered, as well as the factors which 
may cause this to vary. 
Considerable variations are found in the composition 
of urine which has been collected at different times of the 
day. That which is passed immediately after rising may 
differ from that excreted an hour or two after the first 
meal both in the kind and amount of the dissolved solids. 
Sugar and albumin are more commonly excreted after a 
meal, and may be found then, yet not be present in the 
night’s urine. In order to obtain a fair sample for testing, 
the urine should be collected for twenty-four hours and, 
after mixing, a part taken for analysis. In all quantitative 
determinations the volume for twenty-four hours must be 
measured, and when it has been determined how much of 
the substance is present in the portion tested, the amount 
contained in the whole day’s urine should be calculated. 
A statement of the percentage alone has little value if the 
quantity of the urine is not taken into account. To avoid 
fermentation the vessels should be clean and the tests 
should be made as soon as possible. 
The average volume of the urine in twenty-four hours 
is, for an adult, between 1200 cubic centimeters and 1800 
cubic centimeters (40 and GO ounces). This, however, is 
subject to great variations. It is increased by diuretics, by 
diseases, like diabetes and others; it is diminished in febrile 
diseases, in acute nephritis, in some other diseases of the 
kidneys, and usually before the fatal termination of a dis- 
ease. Its variation gives indications of the progress of the THE URINE. 
disease. The volume will be also affected by the amount 
of drink or liquid food and, in general, varies inversely 
with the perspiration. 
From the presence of ferments, the nrine begins to 
undergo a change after it has stood a few hours. The re- 
action becomes alkaline, owing to the production of am- 
monium carbonate from the urea, and this precipitates 
some of the solids, so that the liquid loses its transparency. 
This and other decompositions produce disagreeable odors. 
The odor of normal urine is characteristic. Certain 
foods and medicines change this; e.g., oil of turpentine 
gives an odor of violets. When it putrefies the odor is 
ammoniacal and offensive. In cystitis it is ammoniacal 
when passed. In suppurative diseases the odor ma}'- be 
putrid. 
Fresh, normal urine is clear, but after standing a 
short time a cloud of mucus appears. Pathologically it 
may be cloudy with matters which settle as a sediment. 
They will be discussed under that subject. 
The color of urine is normally some shade of yellow, 
varying from nearly colorless to reddish yellow. The 
former is true of urines containing much water, and the 
latter where the urine is concentrated and of high specific 
gravity. The latter is constant in febrile conditions and 
their severity can here often be judged from the color. 
Pathologically the urine assumes many other shades. 
Presence of blood gives a red or, when methsemoglobin is 
present, a brown. Jaundice gives a greenish cast or 
brownish green; melanotic cancer, almost black; typhus 
or cholera, sometimes blue, from indigo formed by decom- 
position. Some medicinal or poisonous substances change 
the color; thus seuna or rhubarb gives a reddish or brown- 
ish color, which changes to blood red on adding an alkali. 130 
THE HEINE. 
Santonin gives a yellow; carbolic acid and salol a dark 
green to black; antipyrin and quinine often darken it. 
The specific gravity of urine varies with the amount 
of water and dissolved solids. With an increase of the 
water it approaches 1.000, and becomes greater as the 
solids increase. Hence it is easy to ascertain the amount 
of the solids which are present. If the second and third 
decimal figures of the sjoecific gravity are multiplied by 
£.33 it will give very nearly the weight of dissolved sub- 
stances in one thousand parts of urine (grammes per liter). 
Thus, urine of sp. gr. of 1.021 contains about 49 grammes 
in a liter. 
The specific gravity varies under normal conditions 
from 1.002 to 1.030. It is usually between 1.015 and 
1.025. If sugar is not present the variation in specific 
gravity is due almost entirely to that of the urea. Clin- 
ically the specific gravity of urine is determined by an 
hydrometer, called a nrinometer, which consists of a spin- 
die weighted so as to float in pure water at the line marked 
1.000. The specific gravity is indicated by the figures on 
the spindle at the surface of the liquid. TTinometers 
should always be tested in pure water and if they are not 
correct the reading in the urine must be changed to corre- 
spond with the error. Since the specific gravity varies 
with the temperature some standard temperature must be 
adopted. Most instruments are graduated at (10° P. (15.6° 
C.). The urine must be brought to this temperature be- 
fore testing or, if accuracy is desired, the reading corrected 
hy adding 1 in the fourth decimal place for every degree 
Fahrenheit above 60° or subtracting 1 for each degree 
below 60°. In order to obtain accurate results the degrees 
should not be too close together on the spindle. 
The importance of a knowledge of the specific gravity THE URINE. 
131 
is rather to detect marked changes in the urine from a 
series of observations than to he able to infer the presence 
of some abnormal constituent, like glucose, which would 
certainly be found by the subsequent tests. Thus, in 
nephritis a decrease in specific gravity without change in 
the volume indicates that the urea is not being excreted 
and that uraemia may be feared. 
Experiment 268.—Test the accuracy of the urin- 
ometer in water, then take the specific gravity of urine. 
The cylinder must be wide enough for the urinometer to 
float in it without touching. Foam on the liquid, should 
be removed by a piece of filter-paper. 
Experiment 269.-—Test with an accurate urinometer 
the difference in specific gravities of freshly-passed urine 
when at a temperature of from 95° to 98° F. and that at 
60° F. or below. 
The reaction of normal mixed human urine passed 
during twenty-four hours is acid. This acidity is due not to 
a free acid, but to acid sodium phosphate (NaH2PO4) with 
the corresponding potassium salt, and varies in degree at 
different times. The administration of alkaline drugs is 
followed by the urine’s becoming less acid or even alka- 
line. The same effect is produced by vegetable foods. 
These contain the potassium salts of organic acids—citric, 
malic, tartaric, and others—which are oxidized to potas- 
sium carbonate in the system. A similar result is brought 
about a short time after a hearty meal, when hydrochloric 
acid is being set free from its salts in the mucous glands 
of the stomach. The bases which are freed at the same 
time remain to increase the alkalinity of the blood. Part 
of them pass into the urine, producing the “alkaline tide,” 
or alkaline reaction, which is often noticed at this time. 
The urine of herbivorous animals is normally alkaline from THE HEINE. 
this cause. On the other hand, an acid food or one from 
which acids are produced during its decomposition in the 
body will increase the acidity. Such a one is lean meat, 
which contains acid potassium phosphate, and also sul- 
phur and phosphorus compounds, which form sulphuric 
and phosphoric acids by oxidation. Hence the reaction of 
the urine may he to a considerable extent regulated by the 
selection of foods. 
Upon standing all urine becomes alkaline by fermenta- 
tion. This is produced by the action of a number of micro- 
organisms upon the urea, resulting in the formation of 
ammonium carbonate:— 
CO(NU2)2 + 3H20 = (NH4)2CO3. 
If these ferments are introduced into the bladder by 
an unclean catheter the same action is often produced 
there. In chronic inflammation of the urinary tract am- 
monium carbonate is usually present. The latter alka- 
linity—from ammonium carbonate—can be distinguished 
from that produced by sodium and potassium salts by the 
litmus-paper’s resuming its red color after drying, if am- 
monia were present, but not otherwise. 
In determining the degree of acidity of the urine by 
the use of a standard alkaline solution, litmus cannot be 
used to indicate when the neutralization is complete, on 
account of the interference of the phosphates. 
Excessive acidity of the urine causes, in time, an irri- 
tation of the urinary passages, and is favorable to the 
formation of uric acid concretions. Continued alkalinity 
makes a sediment in the urine, and tends to produce phos- 
phatic calculi. It also produces irritation or inflammation 
of the mucous membrane. 
Experiment 270.—Test the reaction of urine with UREA 
133 
sensitive litmus-paper, and if alkaline determine whether 
it is caused by ammonium carbonate by the paper’s turn- 
ing red again after drying, or whether a sodium or potas- 
sium compound is the alkali by the paper’s remaining bine 
on drying. 
Experiment 271. To Determine the Acidity of Urine. 
—To 50 cubic centimeters in a flask add 25 cubic centimeters of 
Vio normal sodium hydrate, and heat to boiling, then remove the 
flame. Thereupon add 25 cubic centimeters of barium chlorid solu- 
tion of about sor 10 per cent. Filter through a dry filter, and take 
50 cubic centimeters of the filtrate, corresponding to 25 cubic centi- 
meters of the urine, for testing. To this add a few drops of phenol- 
phthalein for an indicator, then from a burette, 1/10 normal sul- 
phuric acid until the red color is just destroyed. Subtracting the 
number of cubic centimeters of acid used from 12.5, the number of 
cubic centimeters of standard sodium hydrate in the half of the 
liquid used, gives the number of cubic centimeters of sodium hy- 
drate neutralized by the acids in 25 cubic centimeters of urine. 
Urea. 
About 8G per cent, of the nitrogen in the urine of a 
healthy man has been found to be in the urea, CO(NH2)2. 
Under pathological conditions, however, it may vary greatly 
from this. The absolute weight varies between 30 and 40 
grammes daily, being somewhat less for a woman than for 
a man. In round numbers, we can say that it is about one 
ounce in twenty-four hours for the adult male. 
Urea crystallizes in long, colorless, rhombic prisms. 
It is easily soluble in alcohol and in water; hence it never 
forms a sediment. It forms double compounds with acids, 
some of which, like the nitric and oxalic acid compounds 
are not easily soluble, and are used in separating the urea 
from urine. It forms similar insoluble compounds with 
many salts of the heavy metals, mercury, copper, etc. 134 
THE HEINE. 
When urea is brought into contact with an hypobro- 
mite or an hypochlorite, it is decomposed into carbon 
dioxid, nitrogen, and water:— 
CO(NH)2 + 3NaOBr = SlSTaBr + C02 + N2 + SH,O. 
This decomposition is made use of to determine the 
amount of urea in urine by measuring the volume of the 
nitrogen set free. There are a great number of modifica- 
tions in form of the apparatus employed, Hiifner’s, 
Doremus’s, Squibb’s, and many others,—all based upon the 
same principle. They do not give absolutely accurate re- 
sults, but are sufficiently exact for clinical tests, and have 
the advantage of requiring hut a short time for their exe- 
cution. Where it is desirable to learn accurately the 
amount of nitrogenous compounds excreted, it is best to 
find the total nitrogen by Kjeldahl’s method. The solu- 
tion of sodium hypobromite should be freshly prepared 
from bromin and sodium hydrate, as it decomposes on 
standing. 
Doremus’s ureometer for determining the percentage 
of urea in urine consists of a short graduated tube closed 
at the upper end. Below it is bent upward and expands 
to a bulb. The graduations represent for each division 
0.001 gramme of urea. That is, 0.001 gramme of urea 
evolves enough nitrogen to fill one division. Since one 
cubic centimeter of urine is used, weighing very nearly a 
gramme, nitrogen to fill one division corresponds very 
nearly to 0.1 per cent, of urea in the urine. With the tube 
is furnished a 1-eubic-centimeter dropping pipette. 
An apparatus can he simply and cheaply made, after 
the principle of Squibb’s, from two 4-ounce wide-mouth 
bottles (see figure). One of these (A) contains a vial (O), 
which serves to hold the urine. 135 
AMOUNT OF UREA. 
Outside C, in A, is placed the solution of sodium hy- 
pobromite. B contains water and is connected with Aby 
a rubber tube. When the rubber stoppers are tightly in- 
serted the urine is brought into contact with the hypo- 
bromite by tipping A, the nitrogen of the urea being 
liberated. This forces from B an equal volume of water. 
The water is collected from the tube P, and when meas- 
ured gives the volume of nitrogen set free. The glass 
tubes (I), etc.) should be of small diameter. If there is 
Apparatus for Determining Urea in Urine. 
no leak in the apparatus, pressing the stopper into the 
bottle A will force water from B into the tube P, and it 
should remain full, without running out, as long as the 
apparatus is not disturbed. 
Urea is probably formed in the liver. Its source is 
the nitrogenous compounds of the food and the tissues, 
including tire blood, most of the nitrogen of such com- 
pounds being excreted from the body in the urea. Hence 
any increase in the destruction of these substances is ac- 136 
THE URINE. 
eompanied by an increased formation of urea and vice 
versa. For this reason the urea is considered as a measure 
of the decomposition of the proteins in the body. 
Some things which bring about an increased decom- 
position of proteins are: a large amount of nitrogenous 
food, like meat; excessive exercise, which causes a de- 
struction of tissue, though here the urea is not propor- 
tional to the exertion; fevers and inflammations up to the 
crisis, owing to the rapid loss of muscular tissue. After 
the crisis it is diminished. In phosphorus poisoning and 
diabetes mellitus the urea is excessive for the same reason. 
A greater excretion of water, either from excessive drink- 
ing or diuretics, carries with it a larger amount of urea, 
which seems to be thus washed out of the system. 
On the other hand, less urea is excreted during hunger 
and sleep, when the metabolism of the body is lessened. 
Interference with the excretory power of the kidneys like- 
wise diminishes the urea. This is seen in acute nephritis 
and other diseases of the kidneys. In such cases the pro- 
duction of urea is not stopped, but it accumulates in the 
system, often being accompanied by uraemic poisoning. 
Since the urea is formed, at least in part, in the liver, we 
find that less is excreted in carcinoma and cirrhosis of this 
organ. 
The fermentation of urea to ammonium carbonate, 
caused by the action of micro-organisms, has been already 
referred to. 
PREPARATION OF UREA. 
Experiment 272. From Urine.—lf only a small 
amount is desired, evaporate half a test-tnbefnl of urine 
to dryness on the water-bath. Dissolve the urea from the 
residue with 95-per-cent., alcohol. Allow a drop of the PREPARATION OF UREA. 
alcoholic filtrate to evaporate on a microscope-slide with- 
out the aid of heat. Examine the crystals under the micro- 
scope. If the form is not distinct, dissolve in a drop of 
water and again observe the crystals after this has evapo- 
rated. Add a drop of dilute nitric acid to the slide, let it 
stand a few minutes, then examine the crystals of urea 
nitrate. (Plate I, 6.) 
2. A larger quantity can best he obtained by evapo- 
rating half a liter to a liter of urine to a thin syrup upon 
the water-bath, then cooling it in ice-water, and adding 
about three times its volume of nitric acid of a specific 
gravity of 1.3 which has been boiled to expel the oxids 
of nitrogen and cooled with ice-water. Filter off the 
urea nitrate through an asbestos or glass-wool filter, wash- 
ing with a small quantity of ice-cold concentrated nitric 
acid. Dissolve the crystals in hot water and decolorize by 
ehlorin-water or a small quantity of potassium chlorate. 
Add, then, small portions of pure barium carbonate as long 
as it dissolves and until the liquid is neutral. Evaporate 
the whole upon the water-bath to dryness. Pulverize the 
residue and dissolve the urea in absolute alcohol, which 
does not dissolve the barium nitrate. If the alcoholic solu- 
tion is colored it can be decolorized by filtering it through 
bone-black. Distill off the alcohol or allow it to evaporate 
to obtain the urea. 
Experiment 273. Synthetically.—Coarsely pow- 
der 50 grammes of potassium ferrocyanid and heat in an 
iron dish over a Bunsen flame, stirring continually, until 
it has become a white powder and the lumps show no yel- 
low color when they are broken. If it turns brown the 
heat is too high. Pulverize the mass as finely as possible 
in a mortar, mix it thoroughly with half its weight of 
finely-powdered manganese dioxid, and heat in an iron 138 
THE URINE. 
dish under the hood, stirring meanwhile, until the mass 
glows and becomes thick and sticky. Heat until a small 
test dissolved in hydrochloric acid gives no blue color with 
ferric chloric!. Then allow it to cool; dissolve the potas- 
sium cyanate, which has thus been formed with cold water. 
Convert this into ammonium cyanate by the addition of 
38 grammes of chy ammonium sulphate. Filter and evapo- 
rate upon the water-bath at about 60° to 70°, at which 
temperature ammonium cyanate is converted into urea. 
The potassium sulphate crystallizes out first, and should 
be removed from time to time. At last evaporate to dry- 
ness and dissolve out the urea with absolute alcohol as 
before. 
Experiment 374.-—Mix a few of the dry crystals with 
soda-lime and heat in a dry test-tube. The presence of 
nitrogen is shown by the evolution of ammonia. 
Experiment 375. Preparation oe Sodium Hypo- 
bromite.—In a thin glass flask or beaker containing 80 
cubic centimeters of water dissolve 8 grammes of sodium 
hydrate. Cool, and from a dropping pipette or funnel 
add slowly 3 cubic centimeters of bromin, stirring or 
shaking meanwhile. Handle the bromin under a hood 
or in a draft of air to avoid the vapors, which are espe- 
cially irritating to the eyes and lungs. As the bromin- 
gas is heavy, it should be held below the level of the face 
while pouring, rather than above. 
Experiment 876.—Determine the percentage of urea 
in urine by tbe nse of Doremus’s nreometer. First fill the 
tube with the hypobromite solution and invert it, having 
no more of the liquid in the bulb than is necessary to 
keep the tube full. Fill the pipette exactly to the mark 
with urine, insert the lower end into the nreometer, and 
slowly and steadily force the urine out by compressing 139 
AMOUNT OF UREA. 
the rubber bulb. The urine, being the lighter liquid, 
rises in the nreometer and is immediately decomposed. 
The carbon dioxid is dissolved in the solution and only 
the nitrogen is collected. No gas-bubbles should be 
allowed to escape into the nreometer-bulb or back into 
the pipette, thereby causing a loss. When the foam has 
disappeared, read off the quantity of gas and calculate the 
percentage of urea. Duplicate tests should not differ more 
than 0.1 per cent. If the volume of urine in twenty-four 
hours is known, calculate the. weight of urea excreted in 
that time. 
Experiment 277.—Determine the amount of urea in 
urine by apparatus of Fig. 1. Fill B nearly full of water. 
Into C by a pipette put exactly 2 cubic centimeters of 
urine. Outside of C in A put 20 or 25 cubic centimeters 
of the hypobromite. Insert the stopper (E) tightly, thus 
filling the tidie D with water. If it remains full, showing 
that the apparatus is tight, place an empty beaker under 
I) and gently mix the urine and hypobromite. Avoid as 
much as possible raising the temperature by holding the 
bottle in the hand, as the expansion of the gas causes a 
considerable error. Allow it to stand until no more water 
passes from D, which must remain full of water during the 
whole test; then measure the expelled water in a gradu- 
ated cylinder. 
One gramme of urea contains 371 cubic centimeters 
of nitrogen; but, when it is decomposed in this manner, 
only about 354 cubic centimeters are obtained. For ordi- 
nary clinical purposes the percentage of urea in urine can 
he calculated from the following formula:— 
100 x number of e. c. of N 
percentage of urea 
2x354 THE URINE. 
In more accurate determinations allowance must be made for 
the variation in the volume of the gas from the presence of water- 
vapor, from the variation of the barometer, and the variation in 
temperature from 0° C. This formula is then to be used:— 
W 700 X 354 X u [1 + (0.003665 X t) ] 
W = weight of urea in 100 c. e. of urine, 
v s± volume of water = volume of N in c. e. 
b = height of barometer in mm. 
100 v (b —s) 
s = tension of water at given temperature, 
u = cubic centimeters of urine used, 
t = temperature in degrees centigrade. 
The tension of water-vapor for different temperatures can be 
found in the tables given in any work on gas-analysis. 
Experiment 278. Determination op the Totae Nitro- 
gen in Urine (Kjeldahl’s Method1).-—I. Prepare the following 
solutions:— 
1. Standard sulphuric acid containing about 25 grammes per 
liter, of which the strength has been accurately determined. 
2. Standard ammonia, of which about five volumes are neces- 
sary to neutralize one of the acid. Determine this accurately and 
calculate the amount of ammonia by weight in 1 cubic centimeter. 
3. Potassium sulphid (commercial), about 40 grammes per 
liter. 
4. Sodium hydrate free from ammonia and nitric acid, about 
270 grammes per liter. 
5. Cochineal, made by digesting for a day or two 3 grammes 
powdered cochineal in a mixture of 50 cubic centimeters of alcohol 
and 200 cubic centimeters of water, then filtering. 
Have also at hand:— 
1. Sulphuric acid, sp. gr. 1.84, free from compounds of nitro- 
gen. 
2. Yellow mercuric oxid. 
3. Powdered potassium permanganate. 
11. Operation.—With a pipette measure accurately 5 cubic 
1 This method can be used for finding the amount of N in 
most animal and vegetable compounds. DETERMINATION OE TOTAL NITROGEN. 
141 
centimeters of urine. Place it in a flask holding about 250 cubic 
centimeters, best of hard (Bohemian) glass. Add 0.4 gramme of 
mercuric oxid and 10 cubic centimeters of the concentrated sul- 
phuric acid. Lay the flask in a slanting position on a wire gauze 
over a flame small enough to just bring it to boiling. Perform this 
operation under a hood or where there is a good draft to carry away 
the fumes of the acid. Continue the heating until the liquid is 
colorless or straw-colored, which may require from thirty minutes 
to an hour. Then remove the flask from the flame and very 
slowly add to it a small amount of the powdered permanganate 
until it is colored reddish or greenish. Allow it to cool and pour 
it into an 800-cubic-centimeter flask which contains from 200 to 
300 cubic centimeters of distilled water, rinsing the small flask 
into the large one. The organic matter has been oxidized in this 
process, the nitrogen being converted into ammonia, which is con- 
tained in solution as ammonium sulphate. 
The ammonia is now to be set free by sodium hydrate, then 
distilled into a known amount of standard acid, and its amount 
found by ascertaining the loss of strength of the acid through 
neutralization by the ammonia. For this purpose a Liebig con- 
denser is to be arranged so that the flask can be connected with 
the upper end by means of a bent tube; this should be at least 
1/i inch in diameter inside and a foot long, to prevent small drops 
of the boiling liquid’s being carried over. The insertion of a bulb 
between the flask and condenser, also having the lower end of the 
bent tube, in the flask, cut off obliquely, will aid in preventing this. 
Add, now, to the liquid in the flask a few fragments of granulated 
zinc to make it boil more quietly, 40 cubic centimeters of the 
potassium sulphid to precipitate the mercury, and 80 cubic centi- 
meters of the sodium hydrate, or enough to make it alkaline. 
Connect immediately with the condenser through which a stream 
of cold water is flowing and distill into a 400 or 500 cubic centi- 
meter conical flask (Erlenmeyer) which contains exactly 10 cubic 
centimeters of the standard acid (not the concentrated!). Con- 
tinue the distillation until at least half has been distilled over and 
the distillate coming from the condenser no longer turns red litmus- 
paper blue. 
Then find how much ammonia has been taken up by the 
standard acid. To do this add a few drops of cochineal solution to THE HEINE. 
the distillate. It will be colored yellowish, because of the acid 
reaction. Enough cochineal should be used to give a straw-color. 
From a burette add the standard ammonia, stirring meanwhile, 
until the straw-color just changes to a pink, when the liquid is 
neutral. Subtract the number of cubic centimeters of ammonia 
used from the number which are required to neutralize 10 cubic 
centimeters of the standard acid. The difference represents the 
volume of standard ammonia equal to that which was distilled 
from the oxidized urine. Calculate the weight of NH3 in this. 
Fourteen-seventeenths of the NHa is the weight of the nitrogen 
in 5 cubic centimeters of urine. Calculate the percentage. 
Ueic Acid (“Lithic Acid”). 
Trie acid is normally present in solution in the urine 
of mammals. With birds and snakes it is the principal 
nitrogenous excretory product. Its formula is 
and the composition of the molecule is probably 
HN C NH 'j 
CO C Nil • 
HN CO 
CO 
The daily amount varies much, but averages from 0.3 
to 0.8 gramme. Except that it must he formed from the 
nitrogenous compounds in the body, we know little of its 
production or of the cause and significance of its, variations. 
Uric acid is comparatively insoluble in water or acids, 
but dissolves readily in the fixed alkalies, forming salts of 
uric acid, or urates. In the urine the acid exists in the 
form of these salts or united with some organic base. It 
is a dibasic acid like sulphuric acid, having two atoms of 
hydrogen which can be replaced by metals. It can thus 143 
URIC ACID. 
have two series of salts, the acid and the normal, corre- 
sponding to HKS04 and K2S04. Of these classes the nor- 
mal salts are quite soluble in water, but the acid salts do 
not dissolve so easily. The acid can be set free from its 
salts by the use of a stronger acid. The solubility of the 
acid salts is much less in cold water than in warm. Con- 
sequently they frequently separate from urine which was 
clear when passed but has stood in a cold room, and they 
can then be redissolved by warming. 
When it is pure, uric acid exists in the form of color- 
less crystals. As it is found in the urine, it, as well as its 
salts, is always colored yellow to brown by the coloring 
matter which has been carried down from the urine. The 
simplest form of crystals Is tabular with curved sides and 
pointed ends. These are frequently united at right angles, 
making a star-shaped form, two of the rays often being 
smaller than the other two. In urinary sediments many 
crystals may he united, making a rosette-like form. In 
strongly-acid urine the crystals sometimes have jagged 
edges like the teeth of a broken comb. Many different 
forms may he obtained by precipitating with various 
strengths of acid. (Plate 11, 11.) 
Uric acid and its salts have, in some degree, the power 
of reducing copper compounds in an alkaline solution and 
thus give with Fehling’s test results which are similar to 
those obtained with glucose. When the dry substance is 
warmed with nitric acid it is oxidized, and then gives with 
ammonia a reddish-purple salt, which serves to detect and 
identify the acid. 
The urates as found in the urine are either in solu- 
tion or form a sediment. The latter is generally amor- 
phous and is always colored yellow to brown. Acid sodium 
urate may occur in spherical aggregations of microscopic 144 
THE URINE. 
acicular crystals. Ammonium urate, formed when urine 
becomes alkaline by fermentation, may be found as 
brownish spherules covered with irregular spicules, the 
so-called “thorn-apple” crystals. (Plate 11, 9 and 11.) 
Absolutely accurate methods for determining the amount 
of uric acid in urine are not known. Its amount is some- 
times found by precipitating from a measured volume of 
urine by hydrochloric acid, the albumin having first been 
removed if it is present. After washing the crystals they 
are weighed. The results thus obtained are too low, be- 
cause of the slight solubility of the crystals in water. 
Ahdumetric methods may also be employed. 
Experiment 279.-—Prepare uric acid from urine by 
quite strongly acidifying a beakerful with hydrochloric 
acid. In twenty-four hours the uric acid will have sep- 
arated. Examine the crystals under the microscope. It 
can be purified and gradually freed from the color which 
it derives from the urine by repeatedly dissolving in 
sodium hydrate, diluting with much water and precipi- 
tating again with HCI. 
Experiment 280.—Dissolve a few of the crystals of 
the acid in sodium hydrate and add a few drops of Fehl- 
ing’s solution. Boil and the red cuprous oxid will be 
formed, best seen by the use of a dark background. 
Experiment 281.—To a small quantity of uric acid 
in a porcelain dish add a few drops of dilute nitric acid 
and evaporate to dryness, holding the dish over a small 
flame with the hand in order to avoid heating too highly. 
A reddish-yellow residue is left. Pour into the dish a 
drop of ammonia without at first letting it come directly 
into contact with the residue. In a’short time the residue 
becomes colored reddish purple. The ammonia may be 
added directly to the residue if an excess is not used. An URIC ACID. 
excess destroys the color. The addition of a drop of sodium 
hydrate changes the color to a bluish purple, which is de- 
stroyed on warming. The test is called the murexid test. 
Experiment 282. Preparation of Crystallized Acid 
Urates.—Dissolve uric acid in not a large excess of sodium hy- 
drate, and then pass carhon dioxid into the cold solution until it 
is saturated. Acid sodium urate separates in amorphous masses. 
Experiment 283.—Test the solubility of the acid 
sodium urate by warming with a small quantity of water. 
It will dissolve, and, if not too much water has been used, 
will separate out again when it cools. 
Experiment 284.—Prepare crystallized acid urates by 
dissolving a little uric acid in a warm solution of sodium 
phosphate. Filter, if necessary, and allow the filtrate to 
stand and evaporate. The sodium urate will crystallize as 
masses of acicnlar crystals. 
Experiment 285. Quantitative Determination of Uric 
Acid (Hopkin’s Method).—To 100 cubic centimeters of urine 
add powdered ammonium ehlorid as long as it dissolves, which 
will require about 50 grammes. Let it stand about two hours, when 
the uric acid will he precipitated as ammonium urate. Filter, and 
wash three or four times with a saturated solution of ammonium 
ehlorid. Wash the precipitate from the paper by hot water into a 
small beaker, acidify with HCI, heat to boiling, cool, and allow 
to stand two hours or more. The free uric acid has then crystal- 
lized out. To make correction for the acid which remains dis- 
solved, measure the filtrate before washing and add to the final 
result 1 milligramme for each 15 cubic centimeters. Wash the 
precipitated uric acid on the paper with a little cold water, then 
wash it off from the filter into a flask and warm it with sodium 
carbonate until it has dissolved. Cool and dilute to 100 cubic 
centimeters. Add slowly, while stirring, 20 cubic centimeters of 
pure concentrated sulphuric acid and titrate immediately with one- 
twentieth normal potassium permanganate solution. The latter 
contains 1.578 grammes of pure potassium permanganate in a liter ; 1 
cubic centimeter is decolorized by 0.00375 gramme of uric acid. The HIPPUEIC ACID. 
permanganate solution should be dropped from a burette into the 
uric acid solution until the latter has a pink color, which remains 
a few minutes. The number of cubic centimeters is then read from 
the burette and the amount of uric acid calculated, adding the cor- 
rection for solubility mentioned above. 
Hippuric Acid (C6HbCOHNCH2CO2H). 
This occurs normally in the urine, but in that of 
human beings only in very small quantities. It is found 
here in larger amounts after the internal use of benzoic 
acid. It increases with a vegetable diet and is abundant 
in the urine of herbivorous animals. It forms translucent, 
four-sided prisms, somewhat soluble in water. The acid 
can be made synthetically by heating benzoic anhydrid, 
(C6H5C0)20, with glycocol, NH2CILC02H;— 
(C6H5C0)20 + 2NH2CH2C02H = 2C6HSCONHCH2C02H +h2o. 
When hippuric acid is heated with mineral acids or alka- 
lies it decomposes again into glycocol and benzoic acid. 
Expeeiment 286. Peepaeation of Hippueic Acid. 
—Make the freshly-passed urine of a horse or other 
herbivorous animal strongly alkaline with milk of lime. 
Warm, filter, and evaporate the filtrate to a syrup on the 
water-bath. After it has cooled acidify strongly with con- 
centrated hydrochloric acid. Stir and filter, washing with 
a little very cold water. Dissolve the crystals in the small- 
est possible amount of boiling water. To destroy the color- 
ing matter pass chlorin gas into the hot solution until it 
is light yellow. Then cool it, filter, and wash the crystals 
with a very little cold water. If they are still colored they 
can be still further purified by dissolving in water and 
boiling with a little animal charcoal. Filter, and let the 
acid crystallize from the filtrate. 
Expeeiment 287.—Heat a few of the dry crystals in CHLORIDS. 
a glass tube. They melt and turn red, then give, at first, 
a hay-like odor, afterward the odor of hitter almonds, from 
the hydrocyanic acid formed. On the cooler part of the 
tube is a sublimate of benzoic acid. 
Experiment 388.—On a few crystals in a test-tube 
pour about a cubic centimeter of concentrated nitric acid, 
and bring to a boil. Evaporate to dryness in a porcelain 
dish on a water-bath. The residue, when heated in a dry 
glass tube, gives the odor of bitter almonds. This test can 
be used to detect small quantities of hippuric acid. 
Chlorids. 
In the urine the excreted chlorin, of which there is 
normally in a day 6 to 10 grammes, is united principally 
with sodium. There is a small part with potassium as 
potassium chlorid. The excretion of chlorids in health is 
increased with salt food and with large quantities of drink. 
Chlorids are necessary in the fluids of the body for the 
proper performance of their functions. When more chlorin 
is required by the body the chlorids are held back by the 
kidneys from the urine. When there is a less demand in 
the body the kidneys excrete the chlorids. Thus, in pneu- 
monia and other diseases, where there are serous exuda- 
tions, the chlorids are withdrawn from the circulation to 
form the constituents of these fluids, as is shown by their 
decrease in the urine. When the pathological exudations 
are absorbed the amount of urinary chlorids increases. In 
fevers there is a decrease in the chlorids of the urine until 
the crisis, then an increase. In chronic diseases the amount 
of chlorin gives some indication of the digestive power, 
6 to 10 grammes per day being normal, and less than 5 
grammes daily showing weakness of digestion, providing 148 
THE HEINE. 
that an excessive amount lias not been removed by other 
means, like serous exudations or diarrhoeic discharges. An 
excessive excretion of chlorin (15 to 20 grammes daily) 
is found in diabetes insipidus. In dropsical conditions it 
is a favorable sign, showing the absorption of the fluid. 
The quantity of chlorin can be determined by ascer- 
taining how much silver nitrate is required to precipitate 
it. 
Nad + AgNO.j = AgCl + NaNOy. 
58.4 parts 170 parts 
To ascertain when the chlorin has all united with the 
silver a little yellow potassium chromate is added. The 
silver forms first a white silver chlorid, and when the chlo- 
rin has been precipitated it forms the red silver chromate. 
Expbeiment 289.—Acidify a portion of urine in a 
test-tube with nitric acid and add a little silver nitrate. A 
white precipitate which turns dark in the sunlight indi- 
cates the presence of chlorids. 
Experiment 290. Determination of Quantity of 
Chlorin in Urine.—For clinical purposes the following 
method is sufficiently accurate: Measure with a pipette 
10 cubic centimeters of urine and dilute with about 100 
cubic centimeters of water. Add a few drops of yellow 
potassium chromate solution; then allow to flow into it 
from a burette a solution which contains 17.00 grammes 
of fused silver nitrate in a liter. As soon as the color of 
the precipitate changes from white to reddish, read off 
the volume of silver solution which has been used. Each 
cubic centimeter of this will precipitate 0.00354 gramme 
of chlorin, equal to 0.00584 gramme of sodium chlorid. 
Calculate the percentage of chlorin by weight in the urine. 
The change of color from white to red can be more plainly 
seen by yellow light (gaslight) than by daylight. There PHOSPHATES. 
149 
are present in the urine some other substances which are 
precipitated by silver nitrate like the chlorin. To make 
approximate correction for these, 1 cubic centimeter may 
be subtracted from the number used. 
Phosphates. 
The phosphoric acid of the urine is united with two 
classes of bases: the alkalies,—sodium and potassium,— 
and the alkaline earths,—calcium and magnesium. The 
compounds are called, respectively, “alkaline” and “earthy” 
phosphates. The alkaline phosphates are soluble in water. 
The earthy phosphates are insoluble in water or alkalies, 
but are dissolved by acids. They consequently appear in 
the urine in the insoluble form whenever it becomes alka- 
line, either by fermentation or by the addition of reagents. 
They may also be precipitated by boiling. The amorphous 
white precipitate thus obtained is often mistaken for albu- 
min. It can be distinguished by being easily soluble in 
acids, which is not the case with albumin. When ammonia 
is present, as in fermentation, the magnesium forms an 
insoluble salt with two bases, NH4MgP04. In urinary 
analysis it is referred to as triple phosphate. It is crystal- 
line, sometimes in the form of snow-flakes, but more com- 
monly in prismatic crystals often spoken of as “coffin-lid 
crystals,” from their supposed resemblance to the lid of a 
coffin. (Plate 11, 8.) 
It is the acid phosphates of the alkalies (ISraII2PO4 
and IvH2P04) which give the acid reaction to the urine. 
The phosphoric acid of the urine is mainly that taken 
in the food, but a part comes from the oxidized phos- 
phorus compounds of the tissues, such as lecithin and the 
nuclein compounds. The presence of a sediment of the 150 
THE URINE. 
earthy phosphates shows simply that the urine is alkaline, 
and is no indication that an excessive amount is being ex- 
creted. Animal foods are richer in phosphoric acid com- 
pounds than vegetable; hence with these we find more in 
the urine. 
Experience has shown that there is a diminution of 
the excreted phosphoric acid in many pathological condi- 
tions. This is true in most acute infectious diseases, in 
nephritis, gout, and rheumatism. In diabetes mellitus 
there is an increase. Still, with the exception of the hones, 
the tissues of the body contain hut comparatively small 
amounts of phosphorus compounds, and with our present 
knowledge it is difficult to draw definite conclusions re- 
garding the decomposition of such tissues from the varia- 
tions in the eliminated phosphoric acid. 
Experiment 291.—Make a specimen of urine alkaline 
with sodium hydrate. The earthy phosphates are precipi- 
tated in an amorphous form. Examine under the micro- 
scope. See that they are dissolved again by acidifying with 
even a weak acid, like acetic. 
Experiment 292.—Filter out the earthy phosphates 
and test the filtrate for the phosphoric acid of the alkaline 
phosphates by adding magnesia mixture. (This is mag- 
nesium sulphate made alkaline with ammonia and enough 
ammonium chlorid to dissolve the precipitate first formed.) 
With phosphoric acid it gives a white crystalline precipi- 
tate. 
Experiment 293.—Form triple phosphate by making 
urine faintly alkaline with ammonia and allowing it to 
stand until the precipitate settles. Examine under the 
microscope for the “coffin-lid” crystals. They can be more 
abundantly formed for microscopic examination by adding 151 
PHOSPHATES. 
to the urine a little of a solution of magnesium sulphate 
before making it alkaline with ammonia. 
Experiment 294. Determination of Amount of Phos- 
phoric Acid.-—Prepare the following solutions:— 
1. Uranium acetate: Dissolve about 34 grammes of crystal- 
lized uranium acetate in water and dilute to one liter. This solu- 
tion will be a little too strong. Its exact strength must be found 
by the method to be described later. 
2. A solution of Na2HP04. 12ELO (crystallized disodium phos- 
phate), one liter of which shall contain 10.085 grammes of the pure 
crystallized salt. This salt gives up its water of crystallization 
when exposed to the air, and cannot then be used. The crystals 
must be perfectly bright. Fifty cubic centimeters of the solution 
contain 0.1 gramme of P205 (phosphoric anhydrid). 
3. Solution of sodium acetate of which one liter contains 100 
cubic centimeters of 30-per-cent, acetic acid and 100 grammes of 
sodium acetate. 
4. Solution of cochineal made by digesting for some time 1 
gramme of powdered cochineal in a mixture of 20 volumes of 
alcohol with 60 volumes of water. Filter or decant the liquid. 
Operation.—First ascertain the strength of the uranium solu- 
tion. To accomplish this, measure with a pipette 50 cubic centi- 
meters of the sodium phosphate solution into a beaker; add 5 
cubic centimeters of the sodium acetate solution and a few drops 
of cochineal. Heat to boiling, and then from a burette run in the 
uranium solution, drop by drop, until a greenish color is produced. 
The phosphoric acid has then been precipitated. Since 1 cubic 
centimeter of the uranium solution ought to precipitate 0.005 
gramme of P205, exactly 20 cubic centimeters should have been 
used for the 50 cubic centimeters of sodium phosphate. If this is 
not the quantity which has been used, first ascertain accu- 
rately how much is needed and then dilute the uranium solution 
so that 1 cubic centimeter precipitates 0.005 gramme of P205. If, 
for instance, 17.5 cubic centimeters have been used instead of 20 
cubic centimeters there must be added 2.5 cubic centimeters of 
water for every 17.5 cubic centimeters of the uranium solution. 
The amount of P205 in urine can now he determined in the 
same manner, using urine instead of the sodium phosphate solu- 152 
THE URINE. 
tion. Calculate the percentage of P205 present, knowing that there 
is 0.005 gramme for each cubic centimeter of uranium solution 
which has been used. 
Sulphates. 
The sulphates of the urine are of two classes: (1) 
those of which the base is a metal, like K2S04 and Na2S04, 
and (2) those in which a part or the whole of the base has 
been replaced by an organic radical, like KC6H5S04. 
Those of the first class are called the inorganic, and the 
second the organic, or ethereal, sulphates. The latter dif- 
fer from the inorganic in not forming an insoluble precipi- 
tate upon the addition of a barium salt as the inorganic 
do. The two classes can he separated by this means. After 
the removal of the inorganic sulphuric acid by barium 
chlorid the organic sulphates can be decomposed by means 
of boiling hydrochloric acid:— 
kc6h5so4 + h2o = c 6hsoh + khso4. 
The acid will then give the white precipitate of barium 
sulphate if barium chlorid be added. 
The total amount of combined sulphuric acid excreted 
by an adult in twenty-four hours is 2to 3 grammes. It 
is derived partly from that already formed in the food, 
which passes without change into the urine, but, for the 
most part, from the oxidation of snlphur compounds, like 
albumin, in the body. Variations in the total sulphuric 
acid in general indicate the rate of oxidation of sulphur 
compounds. It is increased by taking such compounds, as 
by a meat diet. It is decreased by a vegetable diet. 
The organic sulphates normally make up about 
one-tenth of the total sulphates. The organic bases of 
these are such compounds as phenol (C0IIr)OTT), eresol SULPHATES. 
153 
(C6H4CII3OH), indoxyl (CBH6NOH), etc. These bases are 
formed by the putrefaction of albuminous substances; con- 
sequently, when such putrefaction is in progress in the 
body the organic sulphates increase in the urine. They 
may be formed in the intestine or absorbed from some 
other source. In the former case they are increased when- 
ever there is a serious stoppage of the food, as in ileus or 
in peritonitis with atony of the intestine. In ordinary con- 
stipation there is no marked increase. In diseases which 
are accompanied by an internal formation of pus there is an 
increased amount of organic sulphates in the urine, and this 
fact may be used to judge whether the pus-forming stage 
has been reached. This is the case in foetid bronchitis, 
carcinoma of the stomach or intestine, diphtheria, pyaemia, 
etc. If the formation is from putrefaction in the intestine 
it will he diminished by taking antiseptic remedies, like 
calomel, or those which, by their purgative action, remove 
the contents of the intestine before this putrefaction has 
occurred. 
The compound of indol which is found in the urine 
goes by the name of indican. The indol, 
C 6H4—CH 
IHST CH 
formed by the putrefaction of albuminous substances, is 
oxidized after it has been absorbed from the intestine or 
elsewhere in the body and becomes indoxyl:— 
CcH-COH 
1 11 
HN CH 154 
THE HEINE. 
This unites with potassium and sulphuric acid to form 
indican;— 
c 6h4—c so4k 
HN CH 
Indican may he easily oxidized by chlorin or other oxidiz- 
ing agents, and then forms indigo blue:— 
C 6H4 —CO CO C 6H4 
1 I i 1 
HN —C = C —NH 
Putrefaction of nitrogenous compounds in the small in- 
testine seems to he more productive of indican than when 
it goes on in the large intestine. Sometimes the indican is 
decomposed in the urine, the indigo being set free in the 
form of blue or red microscopic crystals. It is usually dis- 
solved as a sulphate, however, until the indigo is formed 
by an oxidizing agent. It is normally present in large 
quantities in the urine of the horse, where,* because of the 
long intestine, the residue from the food requires a con- 
siderable time to pass from the body. 
Experiment 295. Preparation op Potassium 
Phenyl Sulphate (KC6HSSO4).—First prepare, if it is 
not at hand, potassium pyrosulphate by mixing 25 
grammes of finely-powdered potassium sulphate with 15 
grammes of concentrated sulphuric acid, then heating 
(best in a platinum dish). The heating should be done 
under a hood, to avoid the acid fumes. The heat should 
be gently applied at first, stirring until all the crystals 
have dissolved. When it ceases to bubble increase the 
heat to low redness. Allow it to cool, but before it solidi- 
fies it is best to carefully pour it upon a piece of clean ORGANIC SULPHATES. 
155 
sheet iron. Powder finely the potassium pyrosnlphate 
(K25207) thus obtained. 
In a thin glass flask holding about a liter dissolve 15 
grammes of potassium hydrate in 20 or 35 cubic centi- 
meters of water, then add 25 grammes of crystallized 
phenol (carbolic acid, C 6H3OH). When it has dissolved 
let it cool to GO0 or 70° C., and, while stirring well, add 
gradually in small quantities 30 grammes of potassium 
pyrosnlphate powdered as finely as possible. Keep it at a 
temperature of from 60° to 70° for from eight to ten 
hours, shaking often. Then add about 125 cubic centi- 
meters of boiling 95-per-cent, alcohol, and filter while it 
is hot. This filtration is best performed in a hot-water 
funnel,—that is, one which is surrounded with a hot-water 
jacket. Otherwise the salt will crystallize out before the 
liquid has passed through the filter. As soon as the filtrate 
cools, the potassium phenyl sulphate crystallizes in pearly 
plates. It should be filtered out and recrystallized from a 
small quantity of boiling alcohol. 
Experiment 296.—Test a solution of this organic 
sulphate with barium chlorid. There is no precipitate. 
Compare the result with that obtained from an inorganic 
sulphate, like magnesium sulphate, with barium chlorid. 
Experiment 297.—Acidify a solution of an organic 
sulphate with hydrochloric acid, boil, and add barium 
chlorid. The acid has decomposed the sulphate; so that 
a precipitate of barium sulphate is now obtained. 
Experiment 298.—Show that a mixture of the two 
classes of sulphates, as in urine, can be detected in this 
way. First acidify by acetic acid, then, after adding 
barium chlorid, let the test-tube stand at least half an 
hour in a beaker of boiling water. The inorganic sulphates 
are thus precipitated as barium sulphate, but not the or- 156 
THE URINE. 
ganic. Filter, and test the filtrate with a drop of barium 
chlorid. If enough was added at first there will be no 
precipitate. If there is, more barium chlorid must he used, 
and the heating repeated. When the filtrate remains clear, 
acidify with hydrochloric acid and boil. The precipitate 
is from the decomposed organic sulphates united with the 
barium chlorid previously added. 
Experiment 299.—Test urine for indican by adding 
to half a test-tubeful an equal volume of concentrated 
HCI. Then add a minute fragment of calcium hypochlo- 
rite (“chlorinated lime”) and a few drops of chloroform 
and shake gently. Let the chloroform settle to the bottom. 
If indican is present in the urine it will he thus oxidized to 
indigo blue, and this colors the chloroform. A second 
jnece of the hypochlorite may be added and the shaking 
repeated. An excess will destroy the blue color. 
Albuminous Compounds oe the Urine 
The principal albuminous substance occurring in the 
urine is serum-albumin. Besides this there may be found 
there serum-globulin, albumose, fibrin, and possibly pep- 
tones. The nucleoalbumins also are not uncommon, being 
often mistaken for mucin. 
albuminuria. 
Serum-albumin may find its way into the urine either 
from the kidneys (renal albuminuria) or from serous 
liquids,—like blood, pus, or lymph,—mixing with it at 
some point in the urinary tract below the kidneys. When 
it is due to degenerative changes in the kidney it is usually 
accompanied by epithelium from the tubules, often in the 
form of cylinders or easts. Changes in the composition ALBUMINURIA. 
157 
of the blood or in the blood-pressure may allow albumin 
to pass through the kidney. This is seen in amende con- 
ditions, after some poisons, and in some infectious dis- 
eases, the kidneys in any of these cases not being neces- 
sarily in a pathological state. Severe muscular labor may 
cause the temporary appearance of albumin. The quantity 
present varies greatly under different conditions, and is 
not necessarily a measure of the severity of the disease. 
Still comparative tests in the same case will indicate some- 
thing of its progress. 
The amount of albumin in the urine can be deter- 
mined accurately by precipitating, drying, and weighing, 
but the process is a long one for clinical purposes. For a 
practical test, sufficient to show the variation in amount, 
Esbach’s method can be used. This depends upon precipi- 
tating the albumin with a solution containing 1-per-cent, 
picric acid and 3-per-eent. citric acid. The operation is 
performed in a graduated test-tube, called an albuminom- 
eter, the height of the precipitate indicating its amount. 
Variations in temperature greatly effect the height of the 
precipitate; consequently in comparative determinations 
the conditions of temperature must he always the same. 
The results are most accurate when not more than 4 
grammes of albumin are contained in a liter. 
TESTS FOE ALBUMIN IN THE HEINE. 
If the urine is not clear it must be filtered before 
testing. 
Experiment 300.—Heat the urine to boiling in a 
test-tube, then acidify with a few drops of concentrated 
nitric acid. If albumin is present, a white precipitate re- 
mains. The earthy phosphates precipitate on boiling, but 
are soluble in acids. 158 
THE URINE. 
Experiment 301.—Pour half an inch of concentrated 
nitric acid into a small test-tube. Hold the tube in a 
slanting position ami slowly pour upon the acid an equal 
volume of urine. If albumin is present a white cloud 
forms at the point of contact of the two liquids. If the 
amount is exceedingly small, it may not appear for half 
an hour. This is Heller’s test. (If biliary pigments are 
present the ring may be colored. See test for these, Ex- 
periment 235.) 
Experiment 302.—Acidify 2 or 3 cubic centimeters 
of potassium ferroeyanid solution with about 1 cubic centi- 
meter of acetic acid, and till the test-tube half full of urine. 
Albumin gives a white, cloudy precipitate. 
Experiment 303.—Add to the urine in a test-tube 
about one-sixth of its volume of a saturated solution of 
sodium chlorid, acidify with acetic acid, and boil the 
upper part of the liquid, holding the tube by the bottom. 
Albumin gives a white precipitate, which shows plainly 
above the clear liquid in the lower part of the tube. 
Each of these tests has some objections to it which must be 
recognized in interpreting the results. By the action of heat and 
nitric acid some of the albumin is decomposed; hence the first test 
is not as sensitive as some others. This decomposition is greatly 
increased if the urine is boiled after adding the acid. Besides 
albumin, there may be precipitated by this test uric acid from the 
urates in very concentrated normal urine, and also resinous matters 
after the administration of turpentine or the balsams. The resin- 
ous compounds are soluble in alcohol, which does not dissolve albu- 
min. The uric-acid compounds are colored instead of being white, 
like albumin, and can be filtered out and tested. 
The ring-test with nitric acid is very sensitive. It precipitates 
other substances than albumin,—such as the urates, mucin, and 
resinous substances. The urates do not form a ring at the plane 
of contact of the two liquids, but above it; and if the urine is 
previously diluted with two or three times its volume of water GLOBULIN. ALBUMOSE. PEPTONE. 
they do not appear. The resinous matters dissolve in alcohol. The 
mucin precipitate forms a cloud in the upper part of the liquid 
where the acid is dilute. It dissolves in strong nitric acid. 
Potassium ferrocyanid and acetic acid will detect very small 
quantities of albumin. Albumose is also precipitated if present. 
If the acid alone produces a cloudiness it is mucin or resinous com- 
pounds. These must be removed by filtration before adding the 
ferrocyanid. 
In the sodium ehlorid and acetic acid test the precipitate 
formed on boiling is acid albumin, which is insoluble in the salt 
solution. Resinous matters may be precipitated, but not mucus. 
Experiment 304. Determination op Amount op 
Albumin in Urine by Esbach’s Method.—The urine 
must not have a specific gravity above 1.008, otherwise it 
must be diluted. If it is not distinctly acid in reaction 
it must he made so hy acetic acid. Fill the albuminometer 
with urine to the mark U. Add the reagent to the mark 
B. Close with a. cork and mix gently, avoiding hard shak- 
ing, which introduces air bubbles into the precipitate and 
thereby prevents its settling. Let it stand at the tempera- 
ture of the room (60° to 70° F.) for twenty-four hours. 
The height of the precipitate indicates the number of 
grammes of albumin per liter, or parts in a thousand. 
globulin, albumose, and peptones. 
Globulin is found in the urine only with albumin. 
It passes into the urine in much the same manner, and 
has no especial diagnostic value. Albumose may be formed 
in urine by bacterial action from albumin. It may easily 
escape discovery, since it is not coagulated by heat. It is 
often the precursor of albumin, and, as such, a knowledge 
of its presence is important. 
The results of the latest research have shown that 
much of what has been regarded as peptone in urine is one THE URINE. 
of the albumoses which closely resembles it, and it is an 
open question whether peptones are ever found in this ex- 
cretion. Nevertheless we may temporarily retain the name 
peptonuria for the condition, with the understanding that, 
as our knowledge becomes greater, it may have to be aban- 
doned. The peptones or albumoses are not normally found 
in the blood, being converted in the intestinal mucous 
membrane into another form, probably into an albumin. 
When anything interferes with this conversion, or when 
they are otherwise introduced into the blood, they pass into 
the urine. Diseases of the intestine, like carcinoma or 
ulceration, may prevent conversion to albumin, giving rise 
to enterogenic peptonuria. Peptone and albumose are 
formed by the decomposition of albuminous substances by 
other means than by digestion; as, for example, by putre- 
faction. Diseases which are characterized by a formation 
of peptones are often accompanied by peptonuria. This is 
the so-called “pyogenic peptonuria.” It is found when 
there is much formation of pus in a body-cavity, as in 
croupous pneumonia and with deep-seated abscesses. Be- 
fore testing for its presence the other albuminous sub- 
stances must be removed. After removing albumin by boil- 
ing the liquid acidified by acetic acid, albumose can be 
detected by its giving a precipitate upon saturation with so- 
dium chlorid, which dissolves on heating and reappears on 
cooling. 
The following tests can be used with urine containing 
a considerable peptone:— 
Experiment 305.—Heat 50 cubic centimeters of urine 
to boiling; acidify if necessary with a few drops of acetic 
acid, filtering if it precipitates, and, while hot, add powdered 
ammonium sulphate to the filtrate as long as it dissolves and 
until there are some crystals in the bottom. Filter after FIBRIN URI A. GLYCOSURIA. 
161 
cooling. This leaves the peptone in solution. To insure 
complete precipitation of the other proteins the saturation 
with ammonium sulphate may have to be repeated. When 
this has been done and no further precipitate results, test 
portions of the filtrate with (1) tannic acid, (2) picric acid, 
and (3) potassio-mercuric iodid for peptone. Each should 
give a yellowish-white precipitate. The biuret test can 
be tried, but is not as sensitive as the others. With pep- 
tones, if no excess of copper sulphate is used, it should 
give a pink with no shade of blue. 
FIBEINURIA. 
Through hasmorrhage or exudation of serous fluids 
into the urinary passages the urine sometimes becomes 
mixed with fibrinogen, and this may form clots or semi- 
gelatinous masses. It may cover the bottom of the vessel 
or occasionally cause the whole mass to gelatinize. The 
fibrin can be filtered from the liquid through muslin and, 
after washing, can be tested (Experiments 67 and 70). It is 
very similar to the deposit of pus from fermenting urine. 
The pus, however, can he thinned with water. The fibrin 
is insoluble. 
GLYCOSURIA. 
Glucose is not normally found in large amounts in 
the urine, although traces are frequently—and perhaps 
always—present. More than a slight trace may be re- 
garded as pathological if it continues for any length of 
time. A transitory form of glycosuria (alimentary gly- 
cosuria) is often caused by excessive quantities of carbo- 
hydrates, especially of sugar in the food. It may be pro- 
duced by puncture of the fourth ■ ventricle of the brain, 
by injuries of the pancreas, by a number of medicinal sub- 162 
THE HEINE. 
stances which act upon the vasomotor nerves of the liver, 
such as phloridzin, etc. 
The urine is generally of a high specific gravity 
(1.030 to 1.050), having a light color and a whey-like 
odor. The daily volume may he increased to ten times 
the normal, the solids being likewise increased. When 
poured or shaken it retains the foam for a considerable 
time. 
Experiment 30G.—Test diabetic urine with 
1. Trommer’s test. 
2. Fehling’s test. 
3. Bottger’s test. 
4. Bismuth subnitrate test. 
5. Phenyl-hydrazin test. 
6. Fermentation test. 
Notice that the other urinary constituents may modify 
the results obtained with the solution of pure glucose. 
For the detection of glucose in urine the tests already given 
may be employed (Experiments 25, 26, 27, 29, 30, and 31). No one 
of these, however, is an absolute proof of the presence of glucose. 
Other constituents of urine have a slight reducing power, and may 
respond to the tests with alkaline solutions of copper or bismuth, 
where the action is that of reduction, for example, uric acid and 
its salts; creatinin, mucin, and others occurring in .smaller 
amounts have this power of reduction. The same is true of many 
medicines which pass into the urine. Trommer’s and Fehling’s 
tests are very sensitive under ordinary conditions, but they may fail 
in some decomposing urines, the ammonia which is present keeping 
the cuprous oxid in solution. Long boiling will expel the am- 
monia, and the test may then succeed. Large amounts of uric acid, 
creatinin, creatin, or albumin may act in the same manner, keeping 
the red oxid from precipitating. 
In the test with the subnitrate of bismuth the salt is not so 
easily reduced by other compounds than glucose. Consequently ACETONURIA. 
there is not so much danger of mistaking these for sugar. With 
a very large excess of the alkali this reduction may occur. This 
is said not to be the ease with Nylander’s modification of the test 
(Experiment 29). Albumin is, however, decomposed under such 
circumstances, giving a black precipitate. It must, therefore, be 
removed from the solution before the test is made. With this test 
very small quantities of sugar can be detected. Many medicinal 
substances pass into the urine and react with this test also. 
The phenyl-hydrazin test is not affected by the reducing 
matters of the urine, but it gives a similar precipitate with milk- 
sugar. Pure phenyl-hydrazin must be used. If it is the hydro- 
chlorid, the crystals should be white, not brown. 
The fermentation test is not very sensitive. It may be inter- 
fered Avith by the presence of some drugs Avhich stop the action 
of the yeast. If the urine is not acid, it should be made faintly 
so with tartaric acid. It can be used to distinguish betAveen glu- 
cose and lactose. Barfoed’s test (Experiment 27) may be employed 
for the same purpose. 
Experiment 307.—Determine the quantity of sugar 
in diabetic urine, using Fehiing’s solution (Experiment 32). 
Dilute the urine with a measured volume of water if 
iiecessar}q and use in the burette, as was done in the case 
of the pure glucose solution. 
Acetone, (CHa)2CO. 
Formally acetone is present in the urine only in 
traces. Pathologically it occurs there in severe diabetes, 
in fevers, in inanition, and cachectic conditions, as well 
as in psychoses. In diabetes it often is a precursor of the 
more dangerous diacetic acid. It appears to be formed by 
the decomposition of albuminous compounds, and it can 
he produced in the urine by the use of a diet of such 
substances. It is a colorless liquid of a fruity odor, which 
boils at 50.5° C. and Avhich can consequently be readily 
distilled from the urine. The examination of the urine 
should he made while it is fresh. 164 
THE HEINE. 
If a large quantity of acetone is present in urine the 
latter may be tested directly. For small amounts it is best 
to distil from about a liter one-fourth of its volume after 
slight acidifying with sulphuric acid. Place the distillate 
in a retort and distil from it about 30 cubic centimeters. 
This latter portion contains most of the acetone. 
Expeeiment 308.—To a solution of acetone add a 
little sodium hydrate, then a solution of iodin in potas- 
sium iodid. lodoform is produced as a yellowish powder 
having a characteristic odor. After a time it may form 
six-sided plates, which can be seen with a microscope. 
Notice also the odor. Alcohol gives the same result. 
Expeeiment 309.—Prepare mercuric oxid by precipi- 
tating a little mercuric chlorid with sodium hydrate. Wash 
by decantation and filter and wash. Add this to some of 
the acetone solution, shake, and filter. The presence of 
acetone is shown by its dissolving the oxid. This can be 
proved by pouring a layer of ammonium sulphid solution 
on top of the filtrate in a narrow test-tube, when the mer- 
cury will be precipitated as a black ring between the two 
liquids. 
Expeeiment 310.—T0 the liquid containing acetone 
add a drop of a freshly-prepared solution of sodium nitro- 
prussid and make alkaline with sodium hydrate. A ruby- 
red color is produced. In a few minutes it changes to 
yelloAV. If it is acidified with acetic acid a carmin or 
purplish-red color appears if much acetone is present. 
On long standing (forty-eight hours) this changes to blue. 
Diacetueia. 
Diacetic or aceto-acetic acid (CH3COCIT2CO2H) 
never appears normally in the urine, but is found under 
the same pathological conditions as acetone. In the fevers DIACETURTA. LACTOSUEIA. 
165 
of childhood it is not so dangerous, but with adults it 
signals the approach of coma, of which it is, perhaps, the 
cause, through lowering the alkalinity of the blood. Di- 
acetic acid is a colorless, strongly-acid liquid, soluble in 
water and ether. On heating it decomposes below 100° 
to acetone and carbon dioxid:—- 
CH3COCH2C02H = CH3COCH3 + COo. 
With ferric chlorid it gives a violet-red solution, which 
disappears on standing twenty-four hours and more quickly 
upon boiling. This reaction can be used to detect diacetic 
acid in the urine. A number of other substances—like 
salicylic and carbolic acids, antipyrin, and the acetates— 
give a somewhat similar reddish color. These are stable 
at ordinary temperatures, and only that from the acetates 
is decomposed by boiling. The test should he made upon 
urine which has been comparatively freshly passed. 
Experiment 311.—Test fresh urine for diacetic acid 
by adding, drop by drop, a solution of ferric chlorid as 
long as a precipitate forms. This is ferric phosphate, 
formed from the phosphates of the urine. Filter, and to 
the filtrate add a few drops more of ferric chlorid. The 
diacetic acid gives a violet-red color. Allow it to stand 
several hours, and notice that it fades and disappears. 
Experiment 312.—If this violet-red color was ob- 
tained, heat another sample of the urine to boiling, and 
after cooling repeat the test. If the red color was caused 
by diacetic acid none will he obtained in this second test, 
since the acid will have been decomposed by boiling. 
Lactosuria. 
Milk-sugar may he found in the urine of women 
toward the end of pregnancy and a short time after child- 166 
THE HEINE. 
birth. Its presence indicates the absorption of the sugar 
from the fluid in the mammary gland. It may appear 
with the interruption of nursing or from stagnation of 
the milk in the gland. When the gland is well developed 
and lactose is found in the urine during the period of 
nursing it shows merely that the secretion of milk is 
abundant. The chemical reactions of lactose are very 
similar to those of glucose. The principal differences are 
that lactose ferments with yeast with difficulty or not at 
all, and that its power of reduction is less than that of 
glucose. Still, the distinction between the two as they 
occur in urine is a matter of some difficulty. 
Expekiment 313.—Try the fermentation test with 
compressed yeast, as in Experiment 31, upon urine con- 
taining glucose and that containing lactose, and notice 
that the former ferments, with the evolution of carbon di- 
oxid, and the latter does not. 
Expeeiment 314.—Try Barfoed’s test (Experiment 
27) upon the two kinds of urine, and notice that it re- 
sponds to glucose, but not to lactose. In this test it must 
be borne in mind that the other reducing substances of 
normal urine—urates, creatinin, etc.—may cause a reduc- 
tion of the copper salt. 
Cholheia. 
In examining the urine for bile two classes of com- 
pounds are sought for: the biliary acids and the biliary 
pigments. The biliary acids do not normally occur in 
urine, except in small amounts. The pigments are more 
commonly found. In the freshly-passed urine usually 
only bilirubin is present, but by oxidation it may be 
generally of a yellowish- to greenish- brown color, and the 
changed to biliverdin, etc. Urine which contains bile is 167 
CTIOLTJRIA. 
sediment, il it contains epithelial cells, is often colored 
brown. Upon shaking the urine the foam is yellow or 
greenish. 
A common cause for the appearance of the biliary 
constituents in the urine is the obstruction of tbe bile- 
duct. This may be either from some abnormal growth 
or merely from inflammation in the passages. The bile 
is then absorbed by the lymphatics and excreted through 
the kidneys. The same result may be produced by any 
abnormal condition of the liver which interferes with the 
free passage of the bile. A part of the bile may pass from 
the blood into the tissues, manifesting itself there by its 
characteristic color (icterus). 
The biliary coloring matters may be formed in tbe liver, but 
they can also be produced by the decomposition of the haemo- 
globin in the blood and the other tissues of the body, and may 
pass from here directly into the urine. In this case the urine 
would contain none of the biliary acids, since they do not appear 
to be formed outside the liver. A large amount of these acids 
with the pigments in the urine indicates that the bile comes from 
the liver (hepatogenous icterus). Some authors have described as 
a distinct form of icterus that where the biliary pigments are de- 
rived from the blood-coloring matters (hsematogenous icterus). It 
seems, however, to be certain that the biliary acids may be absent 
from the urine even when it contains bile from the liver or gall- 
bladder. 
Experiment 315.—Test biliary urine for the pig- 
ments by slowly adding urine to yellow,1 concentrated 
nitric acid in a test-tube held in a slanting position. The 
acid remains in the bottom, and between the liquids are 
seen the colored rings, as in Experiment 236. 
ITlie yellow acid can be made by allowing the colorless acid 
to stand for some time in a strong light. 168 
THE URINE. 
Experiment 316.—1f the urine contains much bilirubin, 
shake a large test-tubeful or more of urine with half an inch of 
chloroform; pour off the urine and let the chloroform evaporate 
on a watch-glass. The bilirubin is left in small, red prisms. It 
may be purified by dissolving in chloroform, filtering, and again 
evaporating. These crystals give the play of colors when moist- 
ened with nitric acid. They also dissolve in alkalies, and the solu- 
tion becomes green on standing (biliverdin). 
Experiment 317.—If the urine is dark colored from much 
urobilin or blood-coloring matters so that the colored rings do not 
show', test it with Huppert’s test. Shake a test-tubeful of the 
urine with a small amount of milk of lime, then immediately pass 
into the liquid a stream of carbon dioxid to remove excess of lime. 
When it is neutral, filter and wash the precipitate, which contains 
the biliary pigments. Moisten the precipitate on the paper with a 
drop of moderately-strong, yellow nitric acid and observe the play 
of colors, from red to green. 
Experiment 318.—1n urine which is highly colored with 
other substances the bilirubin may be identified by Stokvis’s test. 
To 20 or 30 cubic centimeters of urine in a test-tube add 5 or 10 
cubic centimeters of a 20-per-cent, solution of zinc acetate. Wash 
the precipitated bilirubin upon a small filter; then dissolve it by 
the addition of a few drops of ammonia. The liquid which passes 
through the filter becomes, after standing, brownish green, and 
shows the spectrum of bilicyanin: an absorption-band between C 
and D and one between D and E. If much bile is present the 
liquid becomes blue upon slightly acidifying. 
Experiment 319.—T0 the urine add a few drops of 
tincture of iodin. A green color results. If the iodin is 
flowed on to the top of the urine hy slanting the tube a 
green ring is formed. 
Experiment 320.—Test the biliary urine for biliary 
acids hy dissolving in it a few crystals of cane-sugar, then 
dipping in it a strip of filter-paper. Dry the paper and 
place on it a drop of concentrated sulphuric acid. In a 
few seconds it becomes violet,, best seen by holding it be- 
fore a window. Too much sugar gives a brown color. HEMOGLOBINURIA AND HEMATURIA. 
169 
Experiment 321.—Instead of using concentrated acid 
make the test with dilute H2S04, as in Experiment 219. 
It is not advisable to depend upon Pettenkofer’s test alone in 
the urine, as other substances may be present and give reactions 
similar to those of the bile-acids, although their spectra are differ- 
ent. The pure bile-acids may, in-cases of doubt, be extracted by 
the following method:— 
Experiment 322.—If the urine is highly colored or only a 
slight amount of bile-acids are present, it may be necessary to 
extract the latter before testing. Add to the urine lead acetate 
solution and a few drops of ammonia to make it slightly alkaline. 
Wash with water the precipitate, which contains the acids, then 
dry it. Extract it several times with warm alcohol, filtering hot. 
Make the filtrate alkaline with sodium carbonate, and evaporate 
to dryness on a water-bath. Dissolve the sodium salts of the bile- 
acids from the residue with hot, strong alcohol and filter. The bile- 
salts can be precipitated by adding ether to the cooled alcohol. 
They become crystalline on standing, or they can be tested for im- 
mediately in the alcoholic filtrate with Pettenkofer’s or other tests. 
The haemoglobin is found in the urine in two forms: 
first, dissolved, no corpuscles being present (haenjoglohi- 
nnria), and, second, in the corpuscles (haematuria). 
Hemoglobinuria and Hematuria. 
The color of urine which contains blood is usually 
some shade of red, but may be dark brown or even greenish 
brown when the haemoglobin has been changed to met- 
haemoglohin. Very small quantities may not be detected 
by the eye. The liquid is often more or less cloudy from 
corpuscles and casts. There may be enough blood present 
to cause coagulation either in the urinary passages or after 
the urine is passed. 
The free haemoglobin is produced by the destruction 
of the corpuscles. This may he due to an injection of 
substances which dissolve the corpuscles, to the trans- 170 
THE URINE. 
fusion of blood, to the action of some poisons and in cer- 
tain infections diseases, like typhus, also after severe burns. 
In this case the urine should be tested for haemoglobin. 
If there is a sediment the microscope reveals no corpus- 
cles. 
Hsematuria, where corpuscles are present, is more 
common. It is dne to haemorrhage in some part of the 
urinary tract. The corpuscles appear as a sediment and 
are usually not in rolls. They may be shriveled or swollen 
from standing in the urine. If the haemorrhage is from 
the kidney, the blood is usually well mixed with the urine 
and of a reddish-brown color, the reaction being acid. 
Blood-casts may be present, and if they are it is a proof 
of a renal haemorrhage. This may occur in Bright’s dis- 
ease, also with malignant renal growths or renal calculi. 
If the haemorrhage is from the bladder the urine is 
often alkaline, and clots of blood are common. It may be 
caused by vesical calculi, by cystitis or villous growths, and 
by carcinoma. 
Experiment 323.—Add a very little blood to highly- 
colored. normal urine, and notice that the bands of oxy- 
haemoglobin are visible through the spectroscope, although 
to the eye there may be no indication of its presence. 
Experiment 324.—Convert the oxyhaemoglobin into 
haemoglobin as in Experiment 198, and notice that the 
two bands change to one. 
Experiment 325. —To urine containing a small 
amount of blood add enough sodium hydrate to make alka- 
line, and heat to boiling. The phosphates of the alkaline 
earths will be precipitated, and the precipitate will be 
colored reddish by the haematin from the decomposed 
haemoglobin. If no blood were present the precipitated 
phosphates would be Avhite. This test will detect very MUCINURIA. 
171 
small amounts of blood in urine. If tlie liquid is very 
dark colored, it may be necessary to filter and wash the 
precipitate before its color can be determined. 
Experiment 326.—Examine microscopically the sediment 
from a urine after recent haemorrhage. Observe the presence of red 
corpuscles and also the change in their form which takes place 
after standing. 
Mucinueia 
Both normal and pathological urine often contains a 
substance which, although similar to true mucin, yet dif- 
fers from it in many respects. On account of this re- 
semblance it is often called urinary mucin. The latest in- 
vestigations indicate that it is a nucleoalbumin. In nor- 
mal urine it appears after standing as a light, fleecy cloud 
in the middle of the liquid. Its origin is the mucous mem- 
brane,—principally that of the bladder, ureter, and vagina. 
In small amounts it has no special significance. In catar- 
rhal inflammation of the bladder it is abundant. In cys- 
titis and pyelitis it may give the urine a gelatinous appear- 
ance. Mucin is also increased in febrile conditions, as well 
as in nephritis. 
Urinary mucin is precipitated from its solution by 
alcohol or dilute acetic acid without heating. It may be 
precipitated by very dilute mineral acids, but dissolves in 
excess. After precipitation by acids it is soluble in alka- 
lies. Since nucleoalbnmins, like the mucin of urine, are 
composed of an albuminous substance with a nuclein, they 
give most of the reactions of the albumins, such as those 
with potassium ferrocyanid, picric acid, the biuret test, 
etc. Care is necessary, therefore, to avoid confounding 
urinary mucin with small quantites of albumin. They 
can be differentiated by the fact that the mucin is precipi- 172 
THE URINE. 
tatecl in the cold by acetic acid even after the urine has 
been diluted with water, while albumin is not. 
Experiment 327.—Dilute normal urine with its own 
volume of water, acidify a small beakerful with acetic acid 
and allow to stand until the mucin has separated. Filter 
and wash with water. 
Experiment 328.—Show that the mncin dissolves by 
adding a few drops of an alkali, like sodium hydrate, and 
that it is repreeipitated from this solution by acidifying 
again with acetic acid. 
Experiment 329.—1f urine containing much mucin 
can be obtained, apply the general tests for protein arid 
albumin, and notice that it responds to many of them. 
Lipuria, or Chyluria. 
An abnormal condition of the urine—not uncommon among 
the inhabitants of the tropics, but more rare among those of cooler 
climates—is the presence of fat. Lipuria, or the appearance of fat 
in the urine, may be due to an abscess or fatty degeneration of the 
kidney; to an excessive amount of fat in the blood, as in preg- 
nancy; or to conditions which produce fatty degeneration of other 
organs, as the liver, and in phosphorus poisoning, whereby the 
amount of fat in the blood is abnormally increased. The chyluria 
of the tropics is due to the action of a parasite, which causes a 
rupture of the lymph-vessels and allows the lymph to pass into 
the urinary passages. The urine is often milky, and, on standing, 
a creamy layer forms. It contains also the other constituents of 
the lymph, albuminous substances, etc. In cases of lipuria where 
only a small amount of fat is present it may appear in the form 
of drops upon the surface, or it may be present jn microscopic 
globules, either free or in the casts or epithelial cells of the sedi- 
ment. The globules can be perceived with the microscope and 
separated by ether. 
Experiment 330.—Examine microscopically urine containing 
fat. 
Experiment 331.—To half a test-tubeful of urine containing 173 
fat add one-fifth its volume of ether away from the vicinity of a 
flame. Mix by shaking carefully. Allow to stand until the ethereal 
solution of fat rises to the top. Notice that the urine loses its 
milky appearance. Pour off the ether into an evaporating dish and 
let it evaporate without heating. Dip a strip of white paper in the 
residue, and notice that a greasy stain remains after drying. 
URINARY SEDIMENTS. 
URINARY SEDIMENTS. 
Besides the soluble constituents of the urine, there 
are others which appear as an insoluble deposit upon the 
bottom of the containing vessel or floating in the liquid. 
They may he present in the freshly-passed urine or may 
appear after a time. The former are the more important 
to the physician, although some conclusions may he drawn 
from the latter as to the condition of the system. 
For the collection of these sediments the best method 
is by the centrifugal machine, or centrifuge, this requir- 
ing so little time that the examination can be made before 
changes have occurred in any of the constituents. The 
centrifugal machine is essentially an apparatus where 
tubes or other vessels can be set in rapid rotation. These 
tubes swing from their upper end, and as the speed is 
increased assume a horizontal position. The solid con- 
stituents, being heavier than the liquid, are carried by the 
centrifugal force to the bottom of the tube. The tubes 
should contain from 15 to 20 cubic centimeters, and be 
rotated three to live minutes at a speed of at least 1500 
revolutions per minute. 
If a centrifugal apparatus is not at hand, the sedi- 
ment is best collected by allowing the urine to stand in 
a conical glass vessel, containing 4 to 6 flnidounces, until 
it has settled. Then decant the supernatant liquid or 174 
URINARY SEDIMENTS. 
take, by means of a pipette, a sample of the sediment for 
testing. 
Urinary sediments can be divided into two groups: 
the organized—or anatomical—and the unorganized—or 
chemical—sediments. Those of the first group are formed 
by vital processes, and of the latter by chemical force. 
Of the unorganized sediments some are soluble in acid 
and some in alkaline fluids. Their presence depends, 
therefore, upon the reaction of the urine. They fall 
naturally into two classes in accordance with their solu- 
bility, and may be farther subdivided according to their 
microscopic appearance. The table on the opposite page 
gives the most common varieties. 
Before examining the sediment, test with litmus-paper 
the reaction of the urine in which it is found. Then place 
a drop of urine containing the sediment on a glass slide, 
cover with a cover-glass, and examine microscopically 
with a V - or 2/3-inch objective. The microscopic ex- 
amination should be made before the liquid evaporates and 
leaves on the slide the soluble compounds. A higher power 
may be used afterward if necessary, but generally the low 
power is preferable. Chemical reagents may be applied 
on the slide after removing the excess of urine by a piece 
of porous paper. Place one drop of the reagent on the 
slide by the side of the cover-glass. It will flow under 
the cover-glass, and its action can be observed with the 
microscope as it comes in contact with the different sedi- 
ments. Care should be taken not to allow the reagents 
to touch the microscope-stage. If a low power is used 
without a cover-glass these tests may be made in a flat 
watch-crystal. Where large quantities of a reagent are em- 
ployed, as in testing pus with an alkali, the ordinary chem- 
ical vessels are to be used. 175 
CLASSIFICATION. 
Mucus. 
• Epithelium. 
Blood-corpuscles. 
Casts. 
Bacteria. 
Spermatozoa. 
Organized, ok Anatomical. 
Pus. 
Calcium oxalate, 
(envelope shaped). 
Triple phosphate 
(in very weakly 
acid urine). 
Acid calcium 
phosphate. 
Calcium sulphate. 
Tyrosin. 
Crystalline. 
Uric acid. 
Urinary Sediments. 
IN ACID URINE. 
Calcium oxalate 
(dumb-bell). 
Fat. 
Non-crystalline. 
Acid urates. 
Unorganized, ok Chemical. 
Crystalline. 
Triple phosphate. 
Calcium oxalate. 
IN ALKALINE URINE. 
Non-crystalline. 
Earthy 
phosphates. 
Ammonium 
urates. 
Calcium carbonate. 
Calcium oxalate. UKINARY SEDIMENTS. 
Urine containing -pus is turbid when freshly passed, 
and gives the albumin reactions. When much pus is pres- 
ent it soon falls to the bottom as a thick sediment. Small 
quantities may remain suspended for a long time. In urine 
of an acid reaction the pus-corpuscles can be seen. They 
are circular and colorless, about twice the diameter of the 
red blood-corpuscles. They appear granular, but when 
brought in contact with acetic acid the granulation dis- 
appears and the nuclei, of which there are two or three, 
become visible. (Plate 111, 13.) When the urine becomes 
alkaline, either by fermentation or by the addition of a 
fixed alkali, the corpuscles disappear and the mass be- 
comes very sticky and gelatinous, so that it can be drawn 
out by a glass rod into long threads. The turbidity of the 
pustulous urine resembles that from urates or from the 
earthy phosphates. It does not disappear, however, like 
the former, by warming, nor, like the latter, upon tile 
addition of acids. 
The source of the pus in the urine may be anywhere 
in the urinary tract. When it is from the kidney the urine 
is apt to be acid in reaction, and round-celled epithelium 
or casts may be present. When it is from the bladder the 
urine is usually alkaline. It may be due to simple inflam- 
mation or to some deep-seated affection of the tissues. 
Experiment 333.—Examine microscopically urine 
containing pus. Remove the excess of liquid around the 
cover-glass by means of a piece of filter-paper. Put a 
drop of acetic acid on the slide and let it run under the 
cover-glass. Notice the change in the appearance of the 
corpuscles. 
Experiment 333.—Show that the turbidity does not 
disappear upon warming or upon acidifying. 
Experiment 334.—Make Donne’s test for pus by PUS. EPITHELIUM. 
allowing it to settle, then, after decanting off the urine, 
making it alkaline with sodium hydrate. The mass be- 
comes extremely viscid, as is shown by stirring or pouring. 
Experiment 335.—Show that the pus responds to 
the albumin reactions. 
Mucus as a sediment is in the form of a slimy, viscid 
liquid, sometimes showing the mucous corpuscles. Its sig- 
nificance has been explained before. It can he made more 
visible by adding a little tincture of iodin, which colors it 
brownish. The addition of acetic acid to the urine precipi- 
tates mucin as a fibrous mass. 
The epithelial cells, being continually thrown off 
from mucous surfaces, are normally present in small num- 
bers in the urine. In such cases they are usually from the 
bladder and urethra, and, in women, from the vagina. A 
large increase, however, is indicative of a diseased condi- 
tion of some part of the urinary system. The cells from 
different parts of the system are not all of the same shape. 
(Plate 111, 14.) They may be considered as belonging to 
three classes: the squamous, or pavement-epithelium; the 
round celled; and the long, or spindle-celled, epithelium. 
The squamous epithelium is composed of large, fiat, some- 
what irregular cells with a distinct nucleus. They may be 
found singly or united, like the stones of a pavement. 
They occur chiefly in the outer layers of the mucous mem- 
brane of the vagina and bladder. The round-celled epi- 
thelium has smaller cells with a nucleus and nucleolus 
and are found especially in the tubules of the kidneys. 
They are also found in the deeper layers of the mucous 
membrane of other tissues, such as the bladder, urethra, 
and pelvis of the kidney. They are somewhat larger than 
the pus-corpuscles, and the nucleus can be seen without 
clearing by acetic acid. The long-celled epithelium is nar- 178 
ÜBINARY SEDIME N TS. 
row and somewhat irregular, with a nucleus visible with- 
out staining. They are found in the outer layer of the 
membrane of the renal pelvis or in the deep layers of the 
bladder, ureters, and urethra. 
Although the presence of a single kind of epithelial 
cells in the urine may give an indication of their origin, 
still their occurrence in different tissues often renders this 
a matter of doubt. The condition of the cells,- however, 
may furnish information of the pathological changes which 
have taken place. If they appear disintegrated or contain 
fat-globules, their origin is from the locality of some de- 
generative process, often of a chronic nature. 
Blood-corpuscles are not normal in urine. In freshly- 
voided urine they may retain their normal shape,—that of 
a biconcave disk. (Plate 111, 13, d.) In acid urine, espe- 
cially wTere the specific gravity is high, they shrivel after 
a time, the margins becoming irregular. In dilute urine 
and where the reaction is alkaline the corpuscles swell, 
and become biconvex or spherical. If there is much blood 
the liquid is reddish, but a slight amount may escape de- 
tection by the unaided eye. When it is present the albu- 
min reactions can always be obtained. 
By urinary east is meant an irregularly-cylindrical 
mass, composed of various materials, which have been 
formed in the tubules of the kidney, and hence are of 
about the same size as the tubules. Opinions vary as to the 
cause of their formation, but most casts appear to be due 
to the coagulation of the serum which passes into the 
renal vessels owing to some pathological condition. The 
presence of anatomical elements—such as epithelium, pus, 
blood, and fat—or their decomposition products in the 
coagulated mass gives the different varieties of casts. 
Epithelial casts are not very common. They consist CASTS, 
179 
of cylindrical-shaped masses of round epithelial cells which 
are thrown off from the tubules by some pathological proc- 
ess. The cells may appear normal or they may he more 
or less decomposed and of a granular appearance, or they 
may contain minute fat-globules. The cells sometimes 
seem to compose the whole cast and sometimes to he scat- 
tered over its surface. (Plate 111, 16.) When present, 
they indicate inflammation of the kidney. When the cells 
are degenerated, the indications are that the condition is 
chronic or has existed for some time. 
Blood-casts consist of coagulated blood often contain- 
ing so many red corpuscles that they are dark and non- 
transparent. They may he formed whenever haemorrhage 
occurs in the urinary tubules, and are the best evidence of 
this. They are quite rare, and may be obscured under the 
microscope by the free blood-corpuscles. 
Casts of pus are also very rare, but pus-corpuscles 
are not infrequently seen in other varieties of casts. 
By the decomposition and metamorphosis of epithe- 
lium, blood- or pus- cells in casts, the so-called granular 
casts have their origin. They vary greatly in size, shape, 
color, and in fineness of granulation. (Plate 111, 15.) 
The finely granular cannot be easily seen except with a 
high power of the mieroseo|)e, although the coarsely granu- 
lar may be observed with a low degree of magnification. 
They often contain unaltered epithelium, leucocytes, and 
fat-globules. Granular casts indicate degeneration or a 
long-continued pathological condition of the kidney. 
Occasionally casts of fat-globules are observed. They 
result from farther metamorphosis of the granular casts. 
(Plate 111, 18, a.) 
In diseases of the kidney like interstitial suppurative 
nephritis, where bacteria are abundant, casts composed of UEINAIIY SEDIMENTS. 
these organisms are often seen. They resemble granular 
casts, but are not destroyed by mineral acids and caustic 
alkalies, as are the granular casts. High powers of the 
microscope should he used in their examination. 
Hyaline casts are almost transparent or at most show 
only a very fine granulation. On account of their great 
transparency they are extremely difficult to perceive. 
They may be colored yellow by adding a solution of iodin. 
In shape they are usually long and narrow. Besides these 
narrow hyaline casts, which probably are formed in the 
smaller tubules, there is sometimes found a broader vari- 
ety. (Plate 111, 17.) These have an indented edge and, 
in consequence of being more highly refractive, can be 
seen more easily than the narrow ones. They are called 
waxy casts. They often give the amyloid reaction,—a 
brown color with iodin, turning blue to violet upon 
acidifying with sulphuric acid. They are doubtless formed 
in the renal pyramids. The narrow casts dissolve readily 
in acetic acid, but the waxy casts remain in it for some 
time. Hyaline casts not infrequently have anatomical ele- 
ments blood- and pus- corpuscles, epithelium, etc. 
clinging to the surface or included within the mass. 
The origin of the hyaline casts seems to be due to 
the coagulable elements of the blood. It is doubtful if 
they are ever present in urine which has not been albu- 
minous. Their presence, consequently, is indicative of 
the existence of albuminuria. They may be the best evi- 
dence of such a condition, as in interstitial nephritis, 
where the amount of albumin is small. 
Whatever variety of cast may be present in urine, it 
shows, without any doubt, that there is a pathological con- 
dition of the kidney and that the accompanying albumin 
is of renal origin. CASTS 
181 
Besides these cylindrical casts there sometimes appear 
in the urine the so-called cylindroids. These are flat or 
ribbon-shaped, rather than cylindrical. They are usually 
about the diameter of casts, but longer, and resemble in 
their transparency and solubility the hyaline casts, their 
composition being probably the same. They are found in 
nephritis and congestion of the kidneys, also in cystitis. 
They do not seem to be characteristic of any pathological 
condition of the kidneys, but rather of some irritation of 
the lower urinary tract which has extended to the kidneys. 
All casts are decomposed by bacterial action. The 
examination should, therefore, be made as soon as possible 
after the urine is passed and the easts have settled. This 
time may be shortened to five minutes by the use of the 
centrifuge. Without this it will be necessary to let the 
urine stand several hours or over night. 
Experiment 336.—T0 examine urine for casts a few 
drops from the sediment obtained from standing in a con- 
ical glass or from the centrifuge is placed upon the micro- 
scope-slide; one with a shallow cell on top is best. Cover 
it with a cover-glass and remove liquid outside by filter- 
paper. Focus on the sediment, using a 1/5-inch objective, 
then cut off nearly all light from below. When trans- 
parent or hyaline casts are sought for swing the mirror to 
one side and upward and throw the illumination upon the 
slide obliquely or use a small diaphragm. They will be 
more plainly visible by this means than by a strong illu- 
mination. After the casts have been detected their cylin- 
drical shape can be shown by inclining the stage of the 
microscope so that they roll in the liquid. 182 
URINARY SEDIMENTS. 
Bacteria. 
The freshly-voided normal urine contains no bacteria. 
They may be present, however, under abnormal condi- 
tions, and will soon appear in normal urine upon its stand- 
ing exposed to the air. On account of the large amounts 
of organic matter dissolved in the urine, it furnishes a 
medium in which micro-organisms readily grow. This 
occurs even in the bladder if they are introduced from 
the outside, as, for example, by means of an unclean cath- 
eter. Urine containing bacteria is cloudy and is not cleared 
by filtration. 
The non-pathogenic organisms are found in putrefy- 
ing or decomposing urine. This is usually not acid and 
often is strongly ammoniacal. They may be found thus 
in the urine of cystitis where ammoniacal fermentation is 
excessive. Some of these are of large size and can be ob- 
served with a Vg-inch objective without staining. (Plate 
11, 8.) The pathogenic organisms are such as the pus- 
organisms, the diplococcus of gonorrhoea, and also the 
bacillus of tuberculosis and the organisms of infectious 
diseases. They can be examined and isolated by the com- 
mon bacteriological methods. 
Spermatozoa. 
These may be found in the urine of males after coitus 
or pollution. They may be present in some diseases, like 
typhoid, and are constantly found in spermatorrhoea. By 
straining during defecation there may be a slight emis- 
sion of semen, and consequently the spermatozoa be mixed 
with the urine. They are readily recognized by their 
characteristic shape under the microscope,—a flattened oval UEIO ACID AND URATES. 
183 
head united with a long thread-like body and tail. (Plate 
111, 18, c.) They are most abundant in the first and last 
portions of the urine. 
In freshly voided urine they may have some motion, 
but this soon ceases. Acids and alkalies, as well as pure 
water, stop it immediately. Spermatozoa resist putrefac- 
tion and the action of chemical reagents, even that of 
strong acids or alkalies. 
Uric Acid and Urates. 
The properties of these compounds have been given 
before. As a sediment, the free acid and its salts differ 
from all others in being colored yellow to brown. They 
are not abnormal in urine unless they are present as solids 
when the urine is passed, or are deposited within a few 
hours, since normal urine throws down uric acid on fer- 
mentation. The precipitation of these compounds is 
largely effected by a concentration or an increase in the 
acidity of the urine. The normal or dibasic urates are 
readily soluble in water, and do not occur in sediments. 
When the acidity of the liquid is increased, either by 
fermentation or by the addition of an acid, half the base 
is taken from these salts, leaving the monobasic or acid 
urates, which are soluble with much more difficulty. If the 
acidity becomes still greater, all the base is removed, leaving 
the free acid, which is only very slightly soluble in water. 
Of course, a decrease in the volume of water would be ac- 
companied by a corresponding increase in precipitated 
uric acid and its compounds. Hence a sediment of these 
may appear in the urine without signifying that an in- 
creased quantity has been formed in the body. Thus, they 
are common in fevers, when the urine is of small volume 184 
URINARY SEDIMENTS. 
and concentrated. Less uric acid is formed in the body 
with a vegetable diet than with one of meat. 
Uric acid and urates as sediments occur mostly in 
acid urine and can be usually identified microscopically. 
(Plate 11, 11.) The color is characteristic. The acid is 
always crystallized, commonly oval or diamond shaped, 
sometimes visible to the naked eye, often in clusters or 
rosettes. The urates are commonly salts of spdium, potas- 
sium, or ammonium. They may be amorphous when ex- 
amined with high powers. The so-called “brick-dust” 
sediment is a mixture of the sodium and potassium urates. 
Sodium urate is also found in fan-shaped clusters or irregu- 
lar groups of fine crystals, and sometimes in granules. 
(Plate 11, 8.) Ammonium urate makes up the “thorn- 
apple” crystals: brown, spherical masses covered with 
curved spicules. (Plate 11, 9.) The urates can he differ- 
entiated from other sediments by being soluble on gently 
warming the liquid, as well as in alkalies. The urates, as 
well as the free acid, give the murexid test (Experiment 
281). Uric acid is especially important when found as a 
sediment, from its tendency to form calcidi. The same is 
true, to a less extent, of the urates. 
Calcium Oxalate. 
This salt is most frequent in acid urine. It may exist 
in two forms: the crystalline, or “envelope shaped,” and 
the “dumb-bell shaped.” Its appearance under the micro- 
scope affords the best method of identification. (Plate 11, 
10.) The crystalline form consists of octahedral crystals. 
They are never large, often being smaller than a red blood- 
corpuscle. When sufficiently magnified, they have some- 
what the appearance of the hack of a square envelope, the 
crossed lines being formed by the angles of the crystal. CALCIUM OXALATE. PHOSPHATES. 
185 
In the shape of the crystals they resemble some forms of 
triple phosphate, from which they can he distinguished by 
their insolubility in acetic acid and by their smaller size. 
The amorphous form of calcium oxalate is disk shaped, 
with a contraction on opposite sides, so that it somewhat 
resembles a dumb-bell. Calcium carbonate has much the 
same form, hut dissolves in acids with effervescence. 
Calcium oxalate is insoluble in acetic, hut soluble in hydro- 
chloric acid. The dumb-bell form gives rise to calculi of 
the bladder. 
Oxalic acid and its salts are found in many fruits and 
vegetables,—like tomatoes, celery, rhubarb, etc.,—and 
when these are eaten it appears as the calcium salt in 
the urine. It is also produced in the body from certain 
foods,—as from large quantities of nitrogenous foods or 
from the carbohydrates, where the oxidation is not com- 
plete. A small amount, then, may be normal, and if it is 
transitory is of no great consequence. If the excretion is 
continual it is due probably to some constitutional weak- 
ness. 
Phosphates. 
The phosphates of the alkalies, being readily soluble 
in water, do not appear as urinary sediments. The phos- 
phates of calcium and magnesium are insohdde in water 
or alkalies, although they dissolve in acids. They, conse- 
quently, appear as sediments whenever the urine becomes 
alkaline, hut are not found in acid urine unless the acid 
reaction is very faint. They can be distinguished from 
other urinary sediments by dissolving in acetic acid with- 
out effervescence. 
Triple phosphate, NH4MgP04, is a salt of phosphoric 
acid having two bases,—ammonium and magnesium. When 186 
URINARY SEDIMENTS. 
it is made by precipitating a phosphate by ammonia and 
magnesium sulphate the crystals are usually stellate or 
snow-flake formed. As it is made in the urine, however, 
they are more commonly in the form of rhombic prisms. 
The terminations of the prisms are commonly truncated; 
so that the crystals have a shape which approaches that 
of the end of a coffin, and this gives rise to the common 
appellation: “coffin-lid crystals.” (Plate 11, 8.) The 
angles may not be so truncated and the long axis of the 
crystal may be so much shortened that it assumes the form 
of an octahedron, like the calcium oxalate. Unlike the 
latter, it is soluble in acetic acid. Calcium phosphate in 
the urine is usually amorphous, and always colorless. It 
is formed when the urine becomes alkaline in the absence 
of ammonia. To the eye it resembles pus, but differs from 
it in its solubility in acids. In acid urine the acid phos- 
phate, CaHP04, may crystallize in long prisms, usually in 
clusters. Tribasic calcium phosphate, Ca3(P04)2, is color- 
less and amorphous. (Plate 11, 7.) 
The presence of phosphates may be due to an excessive 
formation in the body, and they are then usually accom- 
panied by systemic disturbances. Alkalinity of the urine 
causes their appearance when there is no excess. This may 
be from the food or medicine, from an increase in the alka- 
linity of the blood, or from fermentation. Excessive men- 
tal work is often accompanied by phosphatic sediments. 
Their long-continued presence may excite fear of the 
formation of calculi. Their temporary appearance is a 
matter of no grave significance. In urine which has stood 
for a time after its passage they are the most common of 
the sediments. 
Experiment 337.—Drop ammonia into normal urine 
until it is slightly turbid, and after it has settled examine CALCIUM SULPHATE AND CAE DONATE. 
187 
tlie sediment with the microscope. It is a mixture of the 
amorphous calcium phosphate and crystalline triple phos- 
phate. To obtain a larger amount of the latter add to 
the urine a little magnesium sulphate before it is made 
alkaline. 
Experiment 338.—Precipitate sodium phosphate with 
magnesium sulphate after making alkaline by ammonia. 
Notice the difference in the shape of these crystals under 
the microscope and those formed in the urine. Try the 
solubility of both forms in acetic acid. 
Experiment 339.—Make normal urine alkaline with 
sodium hydrate and examine the precipitated calcium and 
magnesium phosphates with the microscope. Try their 
solubility. 
Calcium Sulphate. 
This does not often occur as a sediment. It may he 
found in acid urines as long prisms united in clusters. 
(Plate 11, 12, a.) 
Experiment 340.—Prepare crystals of calcium sul- 
phate by precipitating a rather dilute solution of calcium 
chlorid with a few drops of sulphuric acid. Dissolve the 
precipitate in boiling water, filtering hot if all does not 
dissolve. It will reprecipitate upon cooling. Examine 
with the microscope. 
Calcium Cardonate. 
This compound is often found in alkaline urine with 
calcium phosphate. It appears as a sandy powder which, 
when examined microscopically, is seen to consist of 
spherical bodies formed of concentric layers or to have 
the dmnh-hell shape of calcium oxalate. (Plate IT, 9.) 188 
URINARY SEDIMENTS. 
It dissolves readily in acetic or other acids, Avith the evolu- 
tion of carbon dioxid gas. 
Tyrosin. 
Tyrosin is not often found as a sediment because of 
its solubility in water, but it sometimes appears as such, 
though never in a normal condition of the system. It 
crystallizes in minute needle-shaped crystals, which are 
usually aggregated into clusters or sheaves. (Plate 11, 
12, c.) Its microscopic appearance is the best means of 
identifying it. The chemical tests have been given. 
Tyrosin in the urine has the same source as in diges- 
tion: the decomposition of protein compounds. It is im- 
probable that it comes from the intestine, but from other 
parts of the system. It is indicative of retrograde met- 
amorphosis of the nitrogenous tissues. Thus, it is present 
in acute atrophy of the liver, in suppurative processes, and 
in phosphorus poisoning, which is accompanied by degen- 
eration of the liver. Leucin is often found at the same 
time. (Plate 11, 12, b.) 
Fat. 
The appearance and significance of fat in the urine 
(lipnria) has already been discussed. 
SYSTEMATIC TESTING OF URINE. 
In the systematic testing of urine the course is often 
varied, as the symptoms may point to the likelihood of 
the presence or absence of certain substances. The quan- 
titative tests may he made use of or not according to 
circumstances. The following are the determinations SYSTEMATIC TESTING OE URINE. 
189 
which are most important, with the tests which may be 
employed:— 
1. Amount passed in twenty-fonr hours. 
2. Color 
3. Transparency 
4. Odor 
Normal or abnormal. 
If the latter, what is the cause? 
5. Chemical reaction. 
If alkaline, is it from NIL or fixed alkalies? 
6. Specific gravity at Go° F. (15.5° C.). 
(Experiment 370). 
7. Urea: quantity, percentage, and amount in twenty- 
four hours (Experiment 376 or 377). 
8. Glucose. 
General test, Trommer’s (Experiment 35). 
Confirmatory tests (Experiments 26, 39, 30, and 
31). 
Quantitative test, Fehling’s (Experiment 34). 
9. Acetone. 
Experiments 308, 309, and 310. 
10. Diacetic acid. 
Experiments 310 and 311. 
11. Albumin. 
General test, heat and HN03 (Experiment 300), 
Confirmatory tests (Experiments 301, 303, and 
303). 
Quantitative test, Esbach’s (Experiment 304). 
13. Blood. 
General test, spectroscope (Experiments 198, 199, 
and 300). 
Confirmatory tests, gnaiacum test (Experiment 
303; also Experiment 385). 
Corpuscles in the sediment. 190 
SYSTEMATIC TESTING OE URINE. 
13. Bile-pigments. 
General test, colors with yellow 11JS103 (Experi- 
ment 315). 
Confirmatory tests (317, 318, and 319). 
14. Bile-acids. 
Experiments 321 and 322. 
15. Peptone. 
Experiment 305. 
IG. Organic sulphates. 
Experiment 298. 
17. Indican. 
Experiment 299. 
18. Uric acid: amount. 
Experiment 285. 
19. Total nitrogen : percentage and amount in twenty- 
four hours. 
Experiment 278. 
20. Chlorin: amount. 
Experiment 290. 
21. Phosphoric acid: amount. 
Experiment 294. 
22. Identification of sediments, if present. 
I. Unorganized. 
{A) Crystalline. 
Uric acid. 
Calcium oxalate. 
Calcium phosphate. 
Triple phosphate. 
Other rarer compounds. 
(B) Amorphous. 
Urates. 
Phosphates, etc. UEI NARY CALCULI 
191 
11. Organized. 
Pus. 
Mucus. 
Blood-corpuscles. 
Bacteria. 
Spermatozoa. 
Epithelium; kind and probable source 
Casts: kind and probable source. 
The proof of the presence of any abnormal constit- 
uent should not be allowed to rest upon one test, but sev- 
eral should be tried. 
The constituents of calculi are the same as those of 
the chemical sediments, and the causes which give rise to 
the formation of the latter will also favor the production 
of calculi in the bladder. To these various names are 
applied, according to their size; sand, gravel, stone, and 
calculi, or concretions. The}'’ vary from the microscopic 
to aggregations as large as an orange. They are generally 
not composed of a single material, but have at the centre a 
nucleus, and this is surrounded by layers, often of two or 
more compounds in alternation. The nucleus may be a 
mass of foreign matter, or it may be a clot of blood or a 
particle of one of the sediments around which material, 
perhaps of a. different kind, can be deposited. Uric acid 
concretions are the most common. They are brown in 
color, rough of surface, and brittle. The form of the 
crystals cannot be seen, but they give the murexid test. 
They dissolve in sodium or potassium hydrate, from which 
solutions tire uric acid may be precipitated in the crystal- 
line form by the addition of a mineral acid. Uric acid 
calculi are formed only in an acid urine. 
URINARY CALCULI. URINARY CALCULI. 
The urates are often found mixed with the uric acid 
deposits or with those of calcium oxalate. The ammonium 
salt is the most abundant. They are generally small,, 
grayish, and rather soft. They give the murexid test. 
They are deposited from acid urine, except the am- 
monium urate, which is formed in an alkaline solution. 
Calcium oxalate concretions are commonly of large 
size and are very hard. The surface is rough and warty. 
They arc called “mulberry calculi” from the resemblance 
of the surface to that of the fruit. The urine is generally 
acid, unless where the presence of the stone has produced 
cystitis. They are often dark in color from the blood which 
has been incorporated with them. 
The phosphates can only be present in calculi when 
the urine is alkaline. They are generally rather soft and 
easily broken. Calcium phosphate has a chalky appear- 
ance. Triple phosphate, NH4MgP04, is found with other 
substances. It is more commonly on the outside of the 
stone, being precipitated by the alkaline reaction produced 
by the presence of the concretion in the bladder. A mixt- 
ure of the triple phosphate and calcium phosphate is fusi- 
ble with the blow-pipe and is known as the “fusible cal- 
culus.” 
Calcium carbonate is not common, although found 
occasionally. 
The analysis of calculi is made by the use of chemical 
methods. The stone should be broken or, better, if it is 
large enough, sawed through the middle. This shows 
the layers of which it is composed and the nucleus. If 
there appears to be any difference in the layers, they should 
be tested separately. Heat a piece upon platinum foil and 
notice udiether it fuses and whether it is combustible or 
not. If it fuses it is an indication of a phosphate of cal- ANALYSIS. 
193 
cium and triple phosphate. If it is combustible it consists 
of organic compounds. 
Blackening when ignited is evidence of organic mat- 
ter, but if slight it may be merely mucus arising from 
irritation of the bladder, and not an essential part of the 
calculus. Ignition on the foil will divide the constituents 
into two classes, although both may be present. 
Combustible, oh 
Organic. 
Incombustible, oh 
Inorganic. 
1. Uric acid. 
2. Ammonium urate. 
1. Calcium phosphate. 
2. Calcium oxalate. 
3. Calcium carbonate. 
4. Triple phosphate. 
5. Urates of K, Na, and Ca. 
If it is composed largely or entirely of organic matter 
try the murexid test (Experiment 281) for uric acid and 
urates. If inorganic compounds are present, powder a piece 
and treat in a test-tube with 2 or 3 cubic centimeters of 
dilute hydrochloric acid. Carbonates dissolve with effer- 
vescence of carbon dioxid gas, the others without. Warm, 
if necessary. Filter, if it does not give a clear solution. 
To one-fourth of the filtrate in a test-tube add sodium 
hydrate until it is alkaline, and test for ammonia by hang- 
ing in the tube a strip of moist red litmus-paper, being 
careful that it does not touch the side of the tube which 
is wet with the sodium hydrate. The tube can be allowed 
to stand corked over night or the ammonia-gas can be ex- 
pelled from the liquid by boiling. If present it will turn 
the paper blue. 
To the remainder of the solution in hydrochloric acid 
add ammonia until it is alkaline, acidify with acetic acid, 
and boil. If there is a precipitate, filter. 194 
UKINARY CALCULI. 
Precipitate is cal- 
cium oxalate. Test 
after washing and 
drying by heating 
to a bright-red heat 
To the filtrate 
boil, and, if there is 
hot. 
add ammonium oxalate, 
a precipitate, filter hile 
A white precipi- 
The filtrate is to be 
on platinum foil. 
tate shows cal- 
tested for magnesium 
After cooling it 
eium, p r o b a b 1 y 
and phosphoric acid. For 
should turn moist 
originally present 
Mg make one-half alka- 
red litmus - paper 
as phosphate or 
line with ammonia and 
blue. 
carbonate. 
if the liquid remains 
clear, add sodium phos- 
phate. A fine, white 
crystalline precipitate 
with either reagent in- 
dicates Mg. For phos- 
phoric acid make re- 
mainder acid with strong 
HN03 and add ammo- 
nium molybdate. A yel- 
low precipitate appears. 
Urates of K, Na, and Ca can be found by boiling the 
powdered calculus in water, filtering, and testing the fil- 
trate by the murexid test. Or if it is evaporated to dryness 
and the residue is ignited on platinum the sodium and 
potassium will remain as carbonates, giving an alkaline re- 
action to litmus-paper. 
THE METEIC SYSTEM. 
In all work in modern chemistry the metric system 
of weights and measures is employed. The unit of length 
is the meter (39.37 inches); of weight is the gramme (or 
gram), which is the weight of 1 cubic centimeter of water 
at -1°; and, of capacity, the liter, which has the volume of 
1 cubic decimeter. THE METRIC SYSTEM. 
195 
Measures oe Length. 
10 millimeters = 1 centimeter. 
10 centimeters = 1 decimeter. 
10 decimeters = 1 meter. 
10 meters = 1 decameter. 
10 decameters = 1 hectometer. 
10 hectometers = 1 kilometer. 
Measures of Weight. 
10 milligrammes = 1 centigramme. 
10 centigrammes = 1 decigramme. 
10 decigrammes = 1 gramme. 
10 grammes = 1 decagramme. 
10 decagrammes = 1 hectogramme. 
10 hectogrammes = 1 kilogramme. 
Measures oe Volume. 
10 milliliters = 1 centiliter, 
10 centiliters = 1 deciliter. 
10 deciliters = 1 liter. 
10 liters = 1 decaliter. 
10 decaliters = 1 hectoliter. 
10 hectoliters = 1 kiloliter. 
One gramme is the weight of 1 cubic centimeter of 
water. 
The following are especially to he remembered:—- 
A liter contains 1000 cubic centimeters and a liter of 
water weighs, therefore, 1000 grammes. 196 
WEIGHTS AND MEASURES. 
The following are convenient in the conversion of the 
weights and measures of one system into another:— 
1 meter = 39.37 inches. 
1 foot = 0.304 meter. 
1 liter =61.03 cubic inches = 1.06 XL S. qts. 
1 liter = 33.81 IT. S. fluidounces. 
1 gramme = 15.43 grains. 
1 grain = 0.0648 gramme. 
1 ounce (apoth.) =31.1 grammes. 
1 ounce (avoirdupois) = 28.35 grammes. 
1 pound (apoth.) = 373.2 grammes. 
1 pound (avoirdupois) = 453.6 grammes. 
REAGENTS. 
E Eli ling’s Solution.—Make up and preserve in two 
parts: A and B. 
(A) Dissolve 34.64 grammes of crystallized, nou- 
effloresced copper sulphate (CuSO4, 5H20) in water and 
make up the volume to 500 cubic centimeters. 
{B) Dissolve 173 grammes of pure, crystallized Ro- 
chelle salt (sodium and potassium tartrate) and 50 grammes 
of sodium hydrate in water, and bring the volume to 500 
cubic centimeters. 
Before using mix equal volumes of A and B. 
Nylander’s Reagent.—Dissolve in 100 cubic centi- 
meters of water 2 grammes of subnitrate of bismuth, 
4 grammes of Rochelle salt, and 10 grammes of NaOH. 
EsbaciTs reagent contains, in a liter, 10 grammes 
of picric acid and 20 grammes of citric acid. 
Millon’s Reagent.—Dissolve 1 part of mercury in REAGENTS. 
197 
3 parts of nitric acid (sp. gr., 1.43), first at ordinary tem- 
perature, then with the aid of heat. When it lias dis- 
solved add twice its volume of water, and after several 
hours decant the reagent from any sediment that may he 
present. 
Barfoed’s reagent contains 0.5 per cent, of acetic 
acid and from 0.5 per cent, to 4 per cent, of cupric acetate 
in water. 
Gttnzberg’s Eeagent for HCI.—Dissolve 3 grammes 
of phloroglncin and 1 gramme of vanillin in 100 cubic 
centimeters of alcohol. 
Boas’s Reagent for HCI.—Dissolve 5 grammes of 
resorcin and 3 grammes of cane-sugar in 100 cubic centi- 
meters of dilute alcohol. 
Methyl-violet 
Congo red 
Tropfeolin 00 
Alizarin sodium 
sulphonate 
A solution in water containing about 1 
per cent, of the coloring matter. 
Phenolphthalein, a 1-per-cent, alcoholic solution. 
Dimethyl-amido-azohcnzol, a 0.5-per-cent, alcoholic 
solution. 
lodin, a 1-per-cent, solution of potassium iodid, in 
water with a few crystals of iodin. 
Of the common reagents, the following strengths may 
be conveniently used:— 
Barium chlorid 
Ammonium hydrate 
Ammonium chlorid 
Tannic acid 
10 per cent, in water, 198 
REAGENTS 
Ammonium oxalate 
Ammonium molyhdate 
Potassium ferrocyanid 
Potass!um ferr!cyanid 
Lead acetate 
Sodium phosphate 
Ferric chlorid 
5 per cent, in water. 
Copper sulphate 
Mercuric chlorid 
Sodium hydrate 
Silver nitrate 
Picric acid 
Lime-water, or calcium hydrate 
A saturated solution in 
water. 
Sulphuric acid, 10 per cent.; pour 1 volume into 18 
volumes of water. 
Nitric acid, 10 per cent.; 1 volume of acid and 6 of 
water. 
Hydrochloric acid, 5 per cent.; 1 volume of acid and 
G of water. 
Acetic acid, G per cent. INDEX. 
Absorption from stomach, test, 
81. 
Acetic acid in stomach, 63. 
Acetone in urine, 163. 
Acid albumin, 39. 
from digestion, 68. 
Acid phosphates in gastric juice, 
64, 72. 
tests for, 73. 
urine, 149. 
Acidity of gastric juice, deter- 
mination, 77. 
urine, determination, 133. 
Acrolein, 22. 
Albuminates, 39. 
Albuminoids, 50. 
Albuminous substances, 30. 
classification, 34. 
in mine, 156. 
Albumins, 35. 
in urine, 156. 
limitations of tests, 158. 
Albuminuria, 150. 
Albumose, 40. 
from digestion, 68, 83. 
in urine, 159, 160. 
preparation, 42. 
Alcohol, production, 13, 15. 
Alkali albumin, 39. 
Alkaline phosphates, 149. 
Ammonium urate, 144. 
Amylopsin, 87. 
Animal body, composition, 1. 
charcoal, 117. 
Anti compounds, 41. 
Bacteria as ferments, 54. 
in urine, 182. 
Bacterial easts, 179. 
Barfoed’s test, 14. 
Blood, 89. 
-casts, 179. 
-coagulation, 90. 
-corpuscles, 89. 
determination of num- 
ber, 90. 
isolation, 92. 
-fibrin, 93. 
in urine, 169, 178. 
-plasma, 89. 
-reaction, 92. 
-serum, 93. 
specific gravity of, 89. 
determination, 92. 
-stains, tests for, 107. 
Bile, 108. 
-acids, 109. 
in urine, 168. 
preparation, 113. 
salts, preparation, 112. 
tests for, 112. 
-pigments, 110. 
in urine, 166. 
preparation, 115. 
tests for, 116, 167, 168. 
Biliary calculi, 111. 
mucin, 108. 
Bilifuscin, 111. 
Biliprasin, 111. 
Bilirubin, 110. 
Biliverdin, 111. 200 
INDEX 
Biuret test, 33. 
Boettger’s test, 14. 
Bone, 116. 
Bone-black, 117. 
Butter-fats, 124. 
Butyric acid, 7!). 
in stomach, 03. 
preparation, 58. 
tests, 79. 
Calcium carbonate in urine, 187. 
oxalate in urine, 184. 
sulphate in urine, 187, 192. 
Calculi of bladder, 191. 
analysis, 193. 
Cane-sugar, 19. 
Carbohydrates, 1. 
classification, 2. 
Carbonic oxid haemoglobin, 101, 
103. 
Carnin, 120. 
Casein, 120. 
preparation, 47. 
Casts in urine, 178. 
bacterial, 179. 
blood, 179. 
epithelial, 178. 
fatty, 179. 
granular, 179. 
hyaline, 180. 
pus, 179. 
waxy, 180. 
Cellulose, 11. 
Cheese, 47. 
Chlorin in urine, 147. 
Cholesterin, 110. 
preparation, 114. 
Cholic acid, 113. 
Choluria, 166. 
Chyluria, 172. 
Chyme, 66. 
Coagulated albumin, 43. 
Coagulation, 31, 37. 
Coffin-lid crystals, 149. 
Collagen, 50. 
Colloid, 4. 
Connective tissue, digestion of. 
65. 
Creatin, 120. 123. 
Creatinin, 120, 123. 
Crystalloid, 4. 
Cylindroids in urine, 181. 
Dextrin, 7. 
Dextrose, 12. 
Diacetie acid, 164. 
Diaceturia, 164. 
Dialysis, 4. 
Digestion, gastric, 65. 
pancreatic, 84. 
salivary, 59. 
Earthy phosphates, 149. 
in urinary sediments, 185. 
Elastin, 53. 
Emulsion, 20. 
Enterogenic peptonuria, 160. 
Enzymes, 54. 
Epithelial casts in urine, 178. 
cells in urine, 177. 
Essential oils, 22. 
Extractives, 119. 
Fat in urine, 172. 
Fats, 20. 
Fatty acids, 22. 
casts, 179. 
Fehling’s test, 14. INDEX 
201 
Fermentation, 54. 
in stomach, 63. 
in urine, 129, 132. 
of glucose, 15. 
Fibrin, 43, 89. 
in urine, 161. 
preparation, 93. 
Fibrinuria, 161. 
Foods, composition of, 1. 
Gall-stones, 111. 
Gastric digestion, 05. 
juice, 02. 
collection, 70. 
preparation of artifi 
cial, 07. 
testing, 71. 
tests, interpretation, 82. 
limitations of, 81. 
Gelatin, 51. 
Globulins, 38. 
in urine, 159. 
Glucose, 12. 
in urine, 101. 
preparation of pure, 13. 
tests, 14, 15, 10. 
in urine, 102. 
limitations, 102. 
Glycerin, formation, 21. 
Glycocholic acid, 109. 
Glycogen, 8. 
Glycosuria, 101. 
Gmelin’s test, 110. 
Granular casts, 179. 
Granulose, 4. 
Grape-sugar, 12. 
Guanin, 120, 122. 
Haematin, 100, 104. 
llamiatogen, 48. 
Haematogenous icterus, 107. 
Haematoporphyrin, 102. 
preparation, 100. 
Hannaturia, 109, 178. 
Hsemin, 100, 104. 
Hscmoel i r ornogen, 102. 
preparation, 107. 
Haemoglobin, 94. 
derivatives, 94. 
quantitative determ ina ti on, 
90. 
spectrum, 95, 102. 
Haemoglobinuria, 109. 
Hainer’s test, 14. 
Heart-burn, 04. 
Hemi compounds, 41. 
Hepatogenous icterus, 107. 
Hippuric acid, 140. 
Huppert’s test, 110. 
Hyalin casts, 180. 
Hydrochloric acid of gastric 
juice, 02. 
tests, 74, 75. 
Hypoxanthin, 120, 122. 
Indican, 154. 
Indigo, 154. 
In dol, 153. 
Indoxyl, 153. 
Inorganic sulphates, 152. 
Invertin, 54, 50. 
Invert-sugar, 19. 
Iron of animal body, source, 48. 
Kephyr, 18. 
preparation, 58. 
Keratin, 53. 202 
Kjehldahl’s determination of ni- 
trogen, 140. 
Koumiss, 18. 
Lactalbumin, 126. 
Lactic acid, fermentation, prep- 
aration, 57. 
in stomach, 63. 
tests, 77. 
sarco-lactie, 123. 
Lactose, 17. 
in urine, 165. 
preparation, 18. 
quantitative test, 127. 
tests, 18, 166. 
Lactosuria, 165. 
Lanolin, 110. 
Lead plaster, 21. 
Lecithin, 26. 
Leucin, from digestion, 83, 85. 
in urine, 188. 
preparation, 87. 
tests, 88. 
Lipuria, 172. 
Lithic acid, d 42. 
Liver-starch, 8. 
Liver-sugar, 8. 
Malt-extract, 121. 
Maltose, 19. 
Malt sugar, 19. 
Methaemoglobin; 99. 
preparation, 106. 
Metric system of weights and 
measures, 194. 
Milk, 124. 
Milk-sugar, 17. 
in urine, 165. 
Millon’s test, 33. 
INDEX 
Mucin, 44. 
in urine, 171, 177. 
Mucinuria, 171. 
Murexid test, 145. 
Muscle, 119. 
-plasma, 119. 
preparation, 121. 
-serum, 121. 
Myelin, 26. 
Myosin, 38. 
Nitrogen, quantitative determ i 
nation, 140. 
Nuclein bases, 49, 120. 
Nucleins, 48. 
Nueleoalbumins, 43. 
Oils, 22. 
Olein, 23. 
Olive-oil, 25, 
Organic sulphates, 152. 
preparation, 154. 
Organized ferments, 54. 
Ossein, 50. 
Oxyhsemoglobin, 98. 
preparation, 105. 
spectrum, 99, 102. 
Palmitin, 22. 
Pancreatic, digestion, 84. 
juice, 82. 
Paranucleins, 48. 
Peptone, 41. 
in urine, 159. 
Pepsin, 64. 
preparation, 56, 66. 
test, 79. 
valuation, 69. 
Pettenkofer’s test, 112. INDEX 
203 
Phenyl-hydrazin test, 15. 
Phosphates in calculi, 192. 
gastric juice, 64, 72. 
urine, 149, 185. 
Phosphoric acid, determination, 
151. 
source, 149. 
Phosphorized fats, 26. 
Plasma, blood-, 89. 
muscle-, 119. 
Proteids, 44. 
Proteins, 28. 
classification, 30. 
Ptyalin, 57, 59. 
preparation, 61. 
Pus-casts, 179. 
in urine, 176. 
Pyogenic peptonuria, 160. 
Reagents, 196. 
Eennin, 65. 
preparation, 68. 
test, 69. 
Rigor mortis, 119. 
Saliva, 58. 
pathological, 60. 
Saponification, 21. 
in digestion, 86. 
Sarein, 120. 
Sediments in urine, 173. 
Sjoqvist’s quantitative test for 
HCI, 73. 
Soaps, 21. 
Sodium hypobromite, prepara- 
tion, 138. 
urate, 143, 145, 184. 
Spermatozoa, 182. 
Starch, 4. 
5, (i. 
Steapsin, 85. 
Stearin, 22. 
Sucrose, 19. 
Sulphates in urine, 152. 
Syntonin, 39. 
Tanning, 51. 
Taurin, 109. 
preparation, 114. 
Taurocholic acid, 109. 
preparation, 113. 
Test-meal, 70. 
Tests, Arnold’s for lactic acid, 
78. 
Barfoed’s for sugar, 14, 18. 
Biuret for albumins, etc., 33. 
Boettger’s for sugar, 14. 
Esbach’s for albumin, 157, 
159. 
Fehling’s for sugar, 14, 10. 
Gmelin’s for bilirubin, 116. 
Guaiacum for blood, 104. 
Haines’s for glucose, 14. 
Huppert’s for bilirubin, 116. 
Kjehldahl’s for nitrogen, 
140. 
Millon’s for proteins, 33. 
Murexid for uric acid, 145. 
Pettenkofer’s for bile acids, 
112. 
Sjbqvist’s for acidity of 
gastric juice, 73. 
Toepfer’s for acidity of gas- 
tric juice, 74. 
Trommcr’s for sugar, 14. 
Xanthoproteic for albumi- 
nous substances, 33. 204 
INDEX 
Toepfer’s test, 74. 
Triple phosphate, 149. 
in sediments, 185. 
Trommer’s test, 14. 
Trypsin, 83. 
Tryptophan, 83, 85. 
Tyrosin, from digestion, 84. 85. 
in urine, 188. 
preparation, 87. 
tests, 88. 
Unorganized ferments, 54. 
urinary sediments, 175. 
Urates, 142. 
in calculi, 192. 
in sediments, 183. 
Urea, 133. 
preparation, from urine, 
136. 
sympathetic, 137. 
quantitative determination, 
138. 
Uric acid, 142. 
in calculi, 191. 
in sediments, 145. 
quantitative determination, 
145. 
Urinary calculi, 191. 
analysis, 193. 
casts, 178. 
mucin, 171, 177. 
sediments, 173. 
Urine, amount, 127. 
color, 129. 
constituents, 127. 
daily variations, 128. 
fermentation, 129. 
odor, 129. 
reaction, 131. 
specific gravity, 130. 
systematic testing, 188, 
Vegetable parchment, 12. 
Waxy casts, 180. 
Xanthin, 120, 122. 
Xanthin bases, 49. 
Xanthoproteic reaction, 33. 
Zymogens, 54.