LABORATORY NOTES 
IN 
PHYSIOLOGICAL CHEMISTRY, 
PART I. 
BY 
FREDERICK G. NOVY, Sc. D., M. D., 
* '• 
Junior Professor of Hygiene and Physiological Chemistry, 
University of Michigan. 
Copyrighted, 1897, by F, Gc Novy. 
F. A. WAGNER, 
Publisher and Mimeographer of Medical Works. 
Ann Arbor, Michigan. P R E P A C E . 
The following notes are temporarily placed in this form in 
order to save much time in dictation and writing. They cover the 
work given in the laboratory during the first four weeks of each 
course and are to be used in connection with the printed "DIRECTIONS"• 
P. G. NOVY. 
March 29, 1897. CONTENTS. 
CHAPTER lo 
PATS 3. 
CHAPTER II. 
CARBOHYDRATES - - 7. 
CHAPTER III* 
PROTEIDS ~ ~ 17 0 
CHAPTER IV. 
SALIVA 30. 
CHAPTER V. 
GASTRIC JUICE 33. 
CHAPTER VI. 
PANCREATIC SECRETION ------ 39. 
CHAPTER VII. 
BILE 45. 
CHAPTER VIII. 
BLOOD 52. 
CHAPTER IX. 
MILK 61. 
CHAPTER X. 
MILK ANALYSIS 66. 3 
CHAPTER I. 
F A T S.. 
PREPARATION OF PURE FAT. 
1) o Cut up lOg of subcutaneous pork fat, or of suet, into as 
small pieces as possible. Place in a small evaporating dish, 
2 1/2-3 inches in diameter, and cautiously heat over a small flame 
stirring continually with- a thermometer. Keep the temperature at 
' 120°~130° * for about 10 minutes. Then strain through a small 
piece of muslin and squeeze thoroughly, reeelvingr-the „clear fat in 
an evaporating dish(4 inch). Transfer the residue to a -small mentor* 
add about 5cc of strong alcohol and rub up fine. Transfer the sus- 
pension now to a 75cc Erlenmeyer flask, rinsing out the mortor with 
several successive small portions of alcohol. Insert into the neck 
of the flask a stopper provided with a condensing tube about 24 
inches "long. Heat on the waterr-bath to boiling for 10 minutes. 
Then set aside and when the suspended particles have settled decant 
the clear alcohol into a small filter and receive the alcoholic 
filtrate in the evaporating dish containing the bulk of the fat. 
To the insolu ble residue remaining in the flask, add 20cc of ether, 
insert condensing tube, and cautiously boil on the water-bath for 
about 5 minutes. Then transfer the entire contents of the flask to 
the filter previously used and receive the ethereal filtrate in the 
evaporating dish. Finally wash the residue with a little ether, 
then squeeze out most of the ether. Open the filter and allow the 
remaining ether to spontaneously evaporate. Save this yellowish 
residue of connective tissue for a subsequent experiment. 
The evaporating dish now contains the strained fat, also the 
alcoholic and ethereal filtrates. Place it on an evaporating dish 
and heat till all the alcohol, ether and water have been driven off 
and only the pure fat remains. 
The student will use for the above experiment, alternately, 
pork fat and suet. 
2) Place a little of the pure fat obtained above on a slide, 
cover and examine under the microscope*- Observe that the little 
round bodies are -compo-sad of crystals. These are more distinct 
in the beef fat. 
3) Transfer a piece of fat, size of a pea, by means of a glass 
rod to a test-tube. Add 5cc of a mixture of equal parts of alcohol 
and ether and warm gently till .dissolved. Then set aside for an 
* The temperature given in the work are Centigrade. FATS. 
4 
hour or more and when a deposit forms transfer some of it by means of 
a pipette to a glass slide, cover and examine under the microscope. 
Sketol, the crystals obtained thus from tallow and from lard. 
Which of these two fats crystallizes most rapidly? 
4) Transfer a piece of fat to a test-tube, add 5cc of alcohol 
and heat till dissolved. Then introduce a strip of blue litmus 
paper, or add a drop of an aqueous solution of litmus. Better still 
to some of the alcoholic solution add a drop of alcholic rosolic 
acid, a yellowish color indicates an acid reaction. What is the 
reaction of normal fat? Why do fats become rancid on standing for 
some time? 
5) . Place a small piece of fat on a filter paper and warm 
gently over a flame, or on a heated plate till the fat melts and 
is absorbed. Note the transparent condition of the paper. 
6) . Rub up thoroughly in a mortor a piece of fat with some 
KHSO4. Transfer the mixture to a dry test-tube and heat cautiously. 
The peculiar irritating odor or sensation is due to acrolein or 
acrylic aldehyde, which is formed by dehydration from the glycerin 
of the fat. 
Glycerin, CH?OH. CHOH.CH OH. 
Acrolein, 
7) To a small piece of fat in the test tube add about lOcc of 
a semi—saturated solution of sodium carbonate, warm and shake thorough 
ly. The liquid becomes milky but on standing-most of the fat col- 
lects on the surface. The liquid below shows but slight cloudiness* 
Neither emulsion, solution or saponification has taken place. 
8) Saponification.--Melt the fat that is left from the pre- 
ceeding experiments and transfer it to a 150cc 'Erlemmeyer flask. 
Then add 2O"30cc of alcohol and 3g of KOH. Insert a condensing 
tube and heat on the wqter—bath for about a half an hour. Sapon—- 
dfication takes place rapidly. To as-ceTtain if the change is com- 
plete pour a little of the alcoholic fluid into a few cc of water*. 
The liquid must remain clear. If it becomes cloudy it is due to 
oil drops and shows that the saponification is incomplete. The 
solution eventually contains soap, glycerine, and excess of alkali 
and alcohol. 
i 
9) Separation of the Fatty Acids.—To about lOOcc of water 
in a small beaker add 3cc of Hf So . Then warm to about 50°. 
Pour the soap solution, gradually, and with constant stirring, into 
the warm acid liquid. The fatty acids are set free and rise to 
the surface forming a clear, oily liquid. Place the beaker on a 
'wate-r~bath and heat till the aqueous liquid below the fatty acid 
* Distilled water is meant in all cases unless otherwise stated. FATS . 
5 
layer becomes almost clear, and all the fatty acid has risen to the 
surface. At the same time prepare some boiling water. 
Then transfer the contents of the beaker to a small filter, 
previously moistened with hot water. The fatty acids, while still 
liquid, are washed on the filter with hot water (10-12 times) till 
the wash water ceases to give with Ball 2 a test for HgSO^. 
Collect the aqueous filtrate and wash-water and set,it aside to be 
examined later for glycerine. 
»phe funnel containing the washed fatty acids is now placed up- 
right in a small beaker containing cold water, the level of which 
should correspond to that of the fatty acid on the filter. The 
fatty acids solidify. The product thus obtained is a mixture of 
oleic acid, C-j 5H34O9, palmitic acid , and stearic acid 
Commercial stearin which is used, in the manufacture 
or candles is a mixture of palmitic and stearic acids. 
10) « Reactions with Patty Acids.--To some of the sokid fatty 
acid in a test-tube add about IGcc of strong alcohol and warm till 
the acid sissolves. Divide into three portions. To one portion 
add a drop of rosolie acid; to another portion 1-2 drops of aqueous 
litmus solution; to a third portion a strip of blue litmus paper. 
What is the reaction and which reagent is more delicate? 
11) To a portion of the fatty acid apply the test given under 
Exp. 5. Why do fats, fatty acids, glycerin water, etc. render 
paper more transparent? 
12) To a portion of the fatty acid apply the test given 
under Exp.6. What is the result? 
13) . To about lOcc of semi-saturated NagCOs solution add a 
small portion of the fatty acids and heat. An effervescence results, 
carbonic acid is given aff, The fatty acids dissolve and a sodium 
soap is formed. Place the tube in a beaker of cold water ,a soap 
jelly results. 
Warm the tube again till the contents are liquid, then add a 
few drops of cottonseed or olive oil and shake. An opalescent 
liquid, or emulsion forms. Transfer a drop of this to a slide, 
cover and examine under the microscope. Note the bighly re- 
fracting fat globules. 
14) Place oome of the fatty acid in a small beaker, add about 
50cc of water and warm gently till the fatty acids mel . Then add 
dilute NaOH- drop by drop, and stir thoroughly after each addition. 
Continue addition of alkali till the fatty acid just dissolves. 
With this sodium soap solution make the following tests: 
a). To some of the solution add a few drops of CaCl2 solution. FATS. 
6 
An insoluble Calcium stearate etc. forms. This calcium soap is 
formed when hard water is used with soap. 
b). To another portion add some lead acetate and warm gently. 
The white sticky precipitate which forms is lead soap. It is 
known and used medicinally as lead plaster. 
15) Separation of Slycerine.--The combined aqueous filtrate and 
washings from the fatty acids, should, if oily globules are present, 
be filtered through a wet filter. The filtrate is then carefully 
neutralized with NaOH and concentrated in an evaporating dish, 
first over a flame, and finally on a water-bath almost to dryness. 
TO the residue add about 25cc of alcohol, stir thoroughly and allow 
the mixture to stand for 1/4-1/2 hour, then filter. To the residue 
add another portion of about 15cc of alcohol, stir well and trans- 
fer this washing to the filter. Evaporate the alcoholic filtrate 
and washings on the water-bath to dryness. Take up the residue 
with about 15cc of absolute alcohol and transfer this entire mix- 
ture to a large test-tube; then add an equal volume of ether 
cork and shake and set aside in a beaker of cold water for about a 
half an hour. Filter off the salts which are thus thrown out of 
solution and cautiously evaporate the alcoholic-ethereal liquid 
on a slightly warmed water-bath. A syrupy residue.* glycerine remains. 
16) Taste the yellowish syrup that is left. 
17) Place a drop of the residue on a slidg and add a little 
powdered borax. Then touch the mixture with a platinum wire and 
place this in a Bunsen flame. Note the green color. 
13). Mix a drop or two of tho syrup with some powdered 
and heat in a dry test-tube. Compare the result with that obtained 
in experiment 6 and 12. 7 
CHAPTER II. 
CARBOHYDRATES* 
In this group are usually placed those substances which contain 
H and. 0 in the same proportion as in water 1.2:1) and 6 carbon atoms 
or a multiple of 6. Recent investigations have shown that we may 
have carbohydrates containing from 4 to 9 or more carbon atoms* 
There are furthermore unquestionable sugars . , as rhamnose, which 
do not have H and 0 in the proportion of 2 to 1* 
Carbohydrates are present in comparatively small amounts* either 
free or as constituentsof certain complex proteids, in the animal 
tody. They constitute, however> the greater part of the solids of 
plants, just as proteids make up the greater part of the animal body* 
They are aldehyde or keton derivatives of certain alcohols* 
The following condensed classification is adapted from ToIIobS* 
I. MONASAC CHAR IDES OR GLYCOSEJ* 
This includes besides others, pentoses, and he*** 
oses, CgHisOft, such as dextrose, laevulose, and rhamnose', 
C6H12°5- 
II. DI - SAC CHAR IDE S, CP. SACCHAROSES, C H 0l;L. 
Cane-sugar, milk sugar, maltose, iob-maltose* 
III. POLYSACCHARIDES* 
A few of these compounds are crystalllzable, but most *f them 
are amorphous. The latter group includes pentosanes, which 
have the same relation in pentoses as starch bears to glucose* 
Also, starch, and its derivitives amylodextrin, erythrodextrin, 
and achrodextrin. Also glycogen, dextran and others, cellulose* 
IV. MANNITS, C ~H-|The compounds of this group are related to 
the true carb^ftyarares. 
v. INOSITBa This is a derivative of haxamet-hyiene G^H-^ 
As shown from the following formulae dextrose or glucose 
contains an aldehyde group whereas laevulose or fructose contains 
a ketone group. 
Dextro s e= CH2OH.CHOH.CHOH.CHOH.CHOH.CHO* 
Laevulo se = CH20H.CHOH.CHOH.CHOH.CO.CHoOH * 
On treatment with nascent hydrogen the aldehyde or ketone 
group is readily reduced to the corresponding alcohol group CH9OH, 
or CHOH* Mazmite CgH 40„, The pentose in a similar way yield 
correspondinf pentites.'' ° CARBOHYDRATES 
8 
The monosaccharides, like aldehydes, readily reduce salts of 
silver, copper, mercury, ect. It should be remembered Lhat other 
substances, as lactose, maltose, glucuronic acid, alkapton, also 
reduce. 
PET1T0S3S, C5Hl05. 
In plants these are substances pentcsaneS, which yield on 
hydration pentoses just as sta oh cn similar treatment yields glucose. 
A pentose has been met with in the decomposition Of glycoprotein 
obtained from the pancreas. It has been found recently in several 
urines} also in the urine of natural and. artificial diabetes* 
The pentoses are strong reducing agents, but are not fermen- 
table by yeasts. With phenylhydraxin they yield osazons which 
melt at 157°-160°. On distillation with hydrochloric acid they 
yield furfurol which colors aniline acetate paper bright red. 
hexoses, c6h12o6. 
Cano-sugar, CgHggOII> yields dextrose and laevulose, 
or hydration can therefore be considered as an anhydride 
of tnese hexoses. The hexoses, dextrose and laevulose, are widely 
distributed in plants, especially in acid fruits; and puthermore 
readily form on hydration of starch, cane-sugar, glncosides as 
phloridzin, etc. Another hexose, galactose, results on hydration 
of lactose, and other carbohydrates, and also of cerebrin. 
The three hexoses mentioned are fermentable by yeast. On 
heating with dilute mineral acids they yield laevulinic acid, 
C5H3O3, humus substances. 
Dextrose or glucose, also known as grape-sugar or starch- 
sugar is, formed during digestion. It is present in small amount, 
0.1-0.2/o, in the blood; in still less amount in normal urine. 
In diabetes it is present sometimes in considerable quantities 
as the characteristic constituent of urine. After the digestion 
of large quantities of sane—sugar, lactose or glucose a reducing 
substance appears in the urine (alimentary glycosuria). A part 
of the cane-sugar may appear as such in the urine. Glucose appears 
in the urine after administration of phloridzin, uranium salts, 
hydrocyanic acid; also the oxygen supply is diminished and 
in GO poisoning. Reducing substances, presumably glucose, and 
formed on the decompost ion of cartilage, nucleinic_ aifcid, paran- 
uclein, nucleoproteid of the pancreas etc. 
It can be obtained as minute crystals which are either anhy- 
drous or contain one molatnAa of water. It is anly about 3/b as 
swee£ as cane-sugar. It is eolxrbla in about an equal part of later} 
insoluble in absolute alcohol. The.solutions are dextro-rotatory. 
The melting-point is at 144-146°; above.200* caramel,forms. carbohydrates 
9 
GLUCOSE. 
f In the following experiments, unless otherwise indicated, 
a 2% solution of glucose Is employed,, 
1) o Molish’s reaction.—To about 1/2 cc of the dilute sugar 
solution add 1-2 drops of an alcoholic solution (15%) of 0(-napthol. 
Then add slowly about lee of concentrated H2SO4 so that it runs 
down the side of the inclined tube and forms a layer. A beautiful 
reddish Triolet ring forms at the zone of contact. 
This is a general reaction due to the formation of furfurol 
and is given by all carbohydrates. Apply this test to some normal 
urine; and to urine diluted with 5 parts of water. If the test- 
is given by the latter it indicates that the carbohydrates of the 
urine are increased. 
2) Place some of the dry glucose in a tube and heat gently 
over a flame. It melts, then turns yellow and finally dark-brown. 
The peculiar odor is that of burnt-sugar. Allow the tube to cool, 
then add water and warm slightly. Note the dark yellow or brown- 
ish color of the solution. Caramel is a harmless coloring matter 
and is employed extensively for coloring liquors, vinegars, etc. 
3) To come dry glucose add cold, concentrated H0SO4 and let 
stand. The liquid remains colorless or at most is light yellow. 
Distinction from cane-sugar. See experiment 3 under cane-sugar. 
After comparing this with the corresponding experiment with cane- 
sugar, gently heat the glucose tube. It promptly blackens due 
to humin substances. Laevulinic acidssformed at the same time . 
4) To the sugar solution add some strong KOH solution and 
heat. The liquid becomes yellow, then dark-brown. The sugar 
undergoes oxidation in alkaline solutions. With solid KOH the 
reaction is sometimes violent because of the heat generated in the 
react ion. 
This test when applied to urine is known as Moore—Heller *s test. 
In that case the precipitate that forms is due to earthy phosphates. 
The test is not particularly delicate and is certainly not reliable 
since the substances may yield dark solutions under similar condi- 
tions, namely alkaptcn, lactose, maltose, etc. 
Compare this reaction with experiment under cane—sugar. 
5) the sugar solution add 1/2 volume of NagCO- solution, 
then 1-2 drops of a freshly prepared solution of potassium ferri— 
cyanide, and boil. yhe liquid becomes colorless—due to a re- 
duction of the silt to a ferrocyanide. 
6) To the sugar solution add a little ammoniacal silver nitrate C A ft fl* d?H V P R' A,? ft 8 
10 
and a few drops of KOH and warm gently. A mirror tf met&lXii 
silver forms, especially if the solutions are dilute. The Silver 
has been reduced. 
The ammcnical silver nitrate is prepared by adding ammonium 
hydrate to the silver nitrate till the precipitate Just appears* 
7) Tc the sugar solution add one drop of 4 freshly prepared 
solution of sodium indigo sulphate, also add a little solU« 
tion and heat. The blue color changes first to violet then to red, 
yellow and finally the liquor is colorless. The indigo has been 
reduced to indigo-white. Cool the tube under the hydrant and shake* 
The indigo—whits is oxidised to indigo blue. On heating again 
the blue is again reduced. Litmus and other coloring agents 
are reduced in a similar manner. 
Tlie following reactions should be applied side bf side, to the 
aqueous solution of glucose and to diabetic urine. 
8) Trornmer’s Test.--Render the solution or urine strongly 
alkaline with KOH and boil then add a few drops of copper sulphate 
solution and warm again a reddish yellow precipitate of cuprous 
oxide forms. If excess of copper be added, the copper hydrate 
precipitate will mask small amounts of the red precipitate. If ttro 
little copper has been added a white precipitate of uric acid 
and nuclein bases (alloxuric bodies) forms. 
8a). Pehling's Test.—Boil some Pehling’s solution in a test- 
tube and then add the sugar solution or the suspected urine and 
boil. Cuprous oxide is thrown down The urine if strongly acid 
should be rendered alkaline. This is the test commonly employed 
when examining for sugar in the urine. It should be remembered 
that it is not an absolute test since the urine, in rare cases, 
may contain other reducing suhstance-s. (alkapt on) • A small amount 
of sugar may, moreover, escape detection since the cuprous oxide 
may be held in solution by creatinin and other urine constituents* 
It sholud furthermore be remembered that Pehling's solution .. - 
deteriorates on keeping, so that on heating the solution itself 
a red precipitate of cuprous oxide may form. It is advisable 
therefore to keep the two constituents of Fehlingfs solution in 
separate bottles and to mix equal volumes just before use. 
Pavy’s solution, employed for the same purpose is a solution 
of copper hydrate in ammonium chloride. 
9) To some Barfoed’s solution add some glucose solution 
and boil* The citprcus oxide precipitate forms. Milk sugar, 
cane-sugar, maltose and. dextrin-do- not reduce this solution. 
reagent is an acid solution of copper. It is pre- 
pared by dissolving l part of copper acetate in 15 parts of water. 
To 200cc of this solution* -of la-added* CARBOHYDRATES 
11 
10) Bottger's Test.—Render the specimen alkaline with sodium 
or potassium hydrate, then add a minute quantity of basic bismuth 
nitrate—-a black color or precipitate due to reduced bismuth, forms. 
Albumin if present must be removed. 
10a). Nylander*s Test.-- Dissolve 10.33g sodium hydrade 
in lOOcc of water; add 2g of basic bismuth nitrate, and 4g Rochelle 
salts; warm and filter; This reagent keeps better that Fehling*s 
solution. 
To 10 volumes of the sugar solution or ufine add one volume 
of the reagent and boil *'.23 minutes. Then let stand for 10-15 
minutes. 
Concentrated urines may become blackish with this reagent; 
if chrysophonic acid is present in the urine this may also occur. 
On the other hand the reaction is more delicate than Fehling’s 
solution, whereas pointed out^small amounts of cuprous oxide 
may be held in solution. 
Alkaline solutions of mercury salts are also employed in test- 
ing for glucose (Knapp, Sachsse). 
11) Phenyl-hydrazin Test.--Phenyl-hydrazin on heating with 
sugar forms phenyl-glycosazon .. ..;« (y/ CgHi 0O4 (N2H. C5H5) p* 
This forms bundles of yellow heedles which melt at 204-205°. 
C 6H12° 6+2C 6H5. N2H3~--C 18H22W|04+2H20+2H. 
Application to the urine.—Place in a small beaker about 50cc 
of the clear urine add 1-2 g of phenylhydrazinc hydrochloride 
and about 2-4 g of sodium acetate, cover with a watch-glass and 
warm on the water-bath for 1/2—1 hour, then turn off the light 
and allow it to cool cn the water-bath. Examine under the micro- 
scope the deposit which forms. If amorphous, or if it is desirable 
to purify the crystals, dissolve on the filter in hot alcohol. 
To the filtrate add water and boil till the alcohol is expelled— 
on cooling the characteristic yellow crystals appear. Filter, 
wash, dry and determine the melting point. 
The phenyl-hydrazin reaction with sugars is of very great 
importance in their identification. It forms with sugars, when 
Seated sufficiently long on the water-bath, ostxzones. The various 
sugars yield therefore corresponding osczones which are yellowish, 
and fdiffer in crystalline form, melting-point, solubility, and 
optical behavior. The determination of the melting-point is es- 
pecially valueable. 
12) Fermentation test.—Rub up some of the solution or of 
the suspected urine with a little yeast. Fill the mixture into 
a large, wide test-tube provided with a perforated stopper through 
which passes a tube bent into a U shape—the free arm being longer 
than the one that passes through the cork. Care should be taken 
to likewise fill the tube so that no air is present in the test-tube CARBOHYDRATES 
12 
when it is inverted. Set the tube adide in an inverted position 
in a warm lace for 24 hours and observe the accumulation of gas* 
When the fermentation is completed place the tube in an up- 
right :ostuion in a dish of water, remove the stopper and by means 
of a bent pipette introduce a little potassium hydrate solution. 
What is the result? 
C ,5**12°6 = 2C2HS°F* + 2CCo. 
Under the influence of certain bacteria it readily undergoes 
lactic acid, lutyric acid or viscous fermentations. 
Laevulose, also known as fruit sugar or frutose occurs, as 
before indicated widely distributed in the plant kingdom. It 
is also present with dextrose in honey. While starch on hydration 
yields dextrose, there are analogous substances, as inulin, 
which on similar decomposition yield laevulose. In ex- 
ceptional cases it has been met with in the urine of diabetes. 
When administered in diabetes a part may be changed to glucose 
and to glycogen, and a part may be eliminated as such (Haycraft). 
This sugar crystallizes with great difficulty and for that 
reason it is ordinarly met with as a thin syrup. It is readily 
soluble in water, insoluble in cold absolute alcohol. The solu- 
tions are laevorotatory. 
The rotation is greater, and in opposite direction, thah that 
of cane-sugar. Hence on hydration of cane-sugar the resulting 
mixture is Laevo—rotatory, and is therefore called invert-sugar-. 
Inversion, as applied to complex carbohydrates, is synonymous 
with hydration. 
Like glucose it reduces readily metallic oxides; is fermented 
by yeast and forms the same osazon. 
Galactose which forms with dextrose on the hydration of milk- 
sugar, and other carbohydrates, also of cerebris. It crystallizes 
in needles or plates which melt at 168°. It is dextro-rotatory. 
It reduces Fehling*s solutiin and is said to ferment with yeast. 
It forma an osazone which melts at 193°, On oxidation it y&el&s 
mucic acid—distinction from dextrose. The origin of galactose 
as a constituent of milk—sugar is not known. It may be derived 
from antecedents in the plant food, and on the other hand may be 
formed from glycogen or even glucose in the body. 
CANE-SUGAR, C 12**22° 11* 
SaccharO&e, Sucrose.- Th'.s sugar is widely distributed in 
plants in the leaves of which, under the influence of light and 
possibly of chlorophyll, it is formed. It is then transported to 
different parts of the plant and may be stored up in the roots as 
in the case of beet root, or in the stalk, as in sugar cane* In 
acid liquid it Very readily undergoes inversion and for that reason 
It is not present in strongly acid fruit juices but ia- represented C A R B 0 H YB1 A T E. S. 
13 
'.here by dextrose and laevulose. In moderately acid fruits as 
nuts, apples, melons, bananas, sweet oranges, it is present as such 
with more or less glucose. The cane-sugar which is removed from the 
flower by the bee becomes almost wholly inverted when made into 
honey0 
It forms large nono-clinic crystals which dissolve in 1*5 
parts of water at 20°, The solution is strongly dextro-rotatory. 
It melts at 160° and bn further heating it yields caramel. It is 
decomposed by dilute acids, in the cold, very rapidly on 
heating. The change is as follows: 
G12H22°11+H2° = CGH12°6 + C6H12°6* 
Dextrose Laevulose. 
This hydration is also brought about Jay many ferments, such as the 
invertin of yeast.; by bacteria and moulds; also by the acid gas - 
trie juice but not by the pancreas. Once inverted the resultant, 
invert-sugar is readily subject to various fermentations such as 
alcohol, viscaas, lactic acid, etc. 
Before inversion it is strongly dextro-rotatory and does not 
reduce Pehling solution, After inversion it is less dextro-rotatory, 
cr even laevo-rotatory, and reduces Pehling solution, With-phenyl 
hydrozin it does not form a corresponding osason, but does form, 
owing to inversion, the phenylglycosazann. This behavior and the 
non-reduction of metallic oxides distinguishes cane-sugar from 
lih&tcso and lactose. The latter therefore still allow the alde- 
hyde chara c te r. 
Apply the following reactions, with the exception of 2 and 3, 
to a 2;f aqueous solution of cane-sugar, and compare these, side by 
side, with the corresponding reactions of glucose. 
1) Molisch’s Reaction.- Apply as given in Ex. 1, under 
glucose * 
2) . Caramel reaction.- Apply as given in Ex. 2, under 
glucose. 
3) Sulphuric acid reaction.- Apply as given in Ex. 3 unds?r 
glucose. The cold acid in a few minutes colors yellow, the n becoms 
black,—distinct ion from Dextrose. Mumin substances are formed. 
4) Potassium hydrate reaction. Apply as given in correo- 
pomding test under glucose and carefully note the difference. 
5) Apply Pehling*s solution as in Ex. 8a under glucose. 
6) o Test’Vith Barford’s reagent, as in Ex. 9 for glucoseo 
7) Tost with Nylander1 s reagent, as in Ex. 10a under glucose. 
8) Apply the fermentation test as in Ex. 12 under glucose 
and compare the rapidity of fermentation with that of glucose. 
9) Place 50 cc. of tje cane-sugar in a small beaker, 
acid 6-8 drops of concentrated H01 and boil for 2-3 minutes. Then 
cool, tender alkaline with sodium or potassium hydrate. To this 
solution now apply tests 4, 5, 6, 7, as given above. Note the results. 
LACTOSE, c12H22°11 + H2°* 
Lactose, cr milk sugar, occurs probably in the mill: of all 
animals* The amount present varies from 3-5—It has been found 
in the urine during the later stages of pregnancy and immediately 
alter birt$n It is said to occur in one plant. 
It forms large rhombic* cyys-taLs. which are soXnbLe in 6 parts 
of cold water and in 2 1/2 parts of boUJLngjffater. .. The solution Is • CARBOHYDRATES. 
14 
dextro—rotatory• When heated to 170—180* it forms lacto carmel, 
mslts at 203.5°• On heating with aeids hydration takes 
plane according to the equation: 
C12H22°11 + H2° “ C6H12°a + a6H120g- 
Oalactose. Dextrose. 
On further heating with acids, humin and formic acid and laevulinic 
acids form. On oxidation with nitric acid inversion first takes 
place as above, and then the galactose is oxidized to mucic acid 
whereas the dextrose forms saccharic acid. It reduces FehlingisoO 
lution but is only 2/3 as strong as dextrose, Unlike the latter it 
is not fermented by yeast. Bacteria readily bring about lactic 
acid fermentation. In kephir and Kumyss the sugar is changed to 
alcohol and lactic acid. 
Lith phenylhydrozin it combines to form a lactosazon which 
crystallizes on cooling as round aggregates of yellow needles which 
melt at 200°« Its behavior to cold concentrated sulphuric acid 
and to alkalies also serves to distinguish it froxn dextrose and 
cane-sugar respectively. Alkalies yield lactic acid and pyroeatechin# 
To a 2/if solution of lactose apply the tests 1-8 as given under 
cane-sugar. For the preparation of milk-sugar see MILK. 
MALTOSE, C12H220i;l * H20. 
This sugar is formed by the action of the gernent diastase, 
contained in malt, on starch. Xt is also formed by the ferments of 
the saliva, pancrea and liver. The formation of dextrin 
precedes that of maltose. When starch is heated with fUSO. maltose 
is temporarily produced. Consequently crude glucose and glucose 
syrup will contain maltose in small amounts. 
It forms fine white needles, grouped in little masses. It is 
soluble in water and in dilute alcohol. On oxidation with nitric 
acid it yields saccharic acid. On heating with sulphuric acid it 
yields two molecules of dextrose. This change is also accomplished 
by ferments. 
Cl2H220l;L + h2o = c6h1206 + c6h12o6. 
Like dextrose it is easily fermented by yeast, and readily reacts 
with potassium hydrate, and with Fehling's soluticn0 It reduces 
the latter more weakly than dextrose, 10 cc..of Fejhling's solution 
represents 77.8 mg. maltose. The reaction with BarfoDd*s reagent 
serves to distinguish it from dextrose. With phenylhydrazin it 
forms an oxazon—Maltozazon. This forms yellow separate needles 
which, melt at 206°. It dissolves or can hold in solution ferric 
hydrate• It is dextro-rotatory. 
Iso-Maltose, is an isomer of maltose. It is amorphous and 
is formed by the action of acids and ferments on starch. It has 
been prepared synthetically from glucose by the action of concen- 
trated H81. Unlike maltose it is more difficultly fermentable, 
and forms an osazon which melts at 153°. It readily reduces 
Fehling*s solution. It is converted by diastase into maltose. 
The relation of the three di-sac char ides can ba from the 
following: 
Cane sugar + HoO = glucose + laovulose. 
a. _L Cl - - - - 2 - •» - - it — CARBOHYDRATES. 
15 
1)o To 100 ec. of boiling water add 10 g„ of starch and stil 
till an even starch pasts forms. Then cool to 60° and add 1 g» of 
powdered, nalt. Immerse in a water-bath at 6Q° for ono hour. At 
intervals of 10 minutes test 1-2 ees of the liquid, with ibdina for 
dextrin (see page ). Then boil and filter. Evaporate the flltrke 
to a thick syrup and wet this adide for several days to crystallize* 
The addition &f a thread, or of a crystal of maltose will favor 
crystallization. Note the taste of the syrup. 
To the remaining 1/2 of the filtrate apply the tests 4-B in- 
clusive as given under cane-sugar. 
STARCH (C6H1005)n* 
Starch, cr amyltim, is a highly complex carbohydrate and the 
value of n in the above formula is not determined. It is placed 
by some at as high as 200. Starches are also known as glucosins 
since on hydration they yield as a final product glucose or dextrose; 
whereas the inulins or laevulans, which correspond to starch, 
yield laevulose. The inulins are comparatively rare, whereas 
starch is a most widely distributed plant constituent« It is evi- 
dently formed from C0o by chlorophyll in the presence of water. 
In plants the excess of sugar as stored up as starch, while in animals 
it is stored up as glycogen* In the body of animals starch can 
unquestionably be converted into and deposited as fats. It is 
known that bacteria acting on starch can give rise to certain fatty 
acids . 
Starch is containedin the so-called starch-granules which have 
a characteristic appearance and can be readily recognized under the 
microscope. The form of the granules as obtained from one plant 
differs from that obtained from other plants, The size of the 
granules varies greatly even in starch of the same variaty. The 
starch proper is deposited in these granules in.layers around one 
or more nuclei. Some cellulose is present. Frequently, as a 
result, concentric rings will be observed in the starch granule. 
On heating to 150-170° it becomes yellowish, and also soluble 
in water; that is, dextrin is formed. Commercial dextrin, which is 
used extensively as a mucilage, is prepared in this way. 
Starch is insoluble in cold water. In the presence of chlo- 
ride of zinc and other salts it swells up and dissolves. On heating 
with water to 60-70° it swells to a pasts but does not form a true 
solution. At a higher temperature it does dissolve forming soluble 
starch and hydrolytic produces described below. With glycerin, 
especially on heating, it forms soluble starch. On heating with 
dilute acids it dissolves readily, or is rather hydrated forming 
soluble products. The final product of the action of an acid is 
dextrose. HC1 acts more rapidly than Diastatic enzymes, 
such as are contained in malt, saliva, pancreas, dissolve starch 
forming a number of intermediate products and finally maltose, 
not dextrose. Starch is not fermented by yeast but is affected by 
bacteria, such as lactic acid and butyric acid bacilli;also by 
moulds. Nitric acid first inverts starch, then oxidizes ...the 
products of saccharic, tartaric, and oxalic acids. 
The products formed, by the hydration of starch, brought about 
by water under pressure, by acids, or by ferments, are presented 
in the following table: carbohydrates. 
16 
Stareh 
iodine, 
blue. 
Fehling, 
-0 
Tastelas8 
Soluble Staroh • 
« 
if 
w 
-0 
ft 
l$-;dzr±tLr i n) 
w 
red 
" + 
very slight 
f! 
• 
Aehroodexbrin 
H 
:o 
n 
+ slight 
ft 
Maltodextrin 
rt 
<. O 
n 
Sweetish* 
Isomaltose 
n 
0 
n 
+ 
Sv/eet« 
Maltese 
h 
0 
n 
+ Barfoec.1,0 
ti 
Dextrose 
h 
0 
n 
ft 
1) Examine microscopically and. sketch the granules of the 
following starches: potato, wheat, buck-wheat, corn, arrow-root 
and rice* Note the shape of granules, the number of rings if any# 
and the eldft or hilum. 
2) * Place a little starch in a test-tube add water and shake 
thoroughly, then filter, To the filtrate add a drop of iodine 
solution. No color is formed, since starch is insoluble* Add 
a drop or two of iodine to the residue on the filter—a blue color 
results. 
3) Soluble starch.—Place lOOcc of water in a beaker and boil 
then add lg of powdered starch and co ntinue boiling for 2-3 minutes 
stirring constantly. A starch paste forms. 
4) o Place some of the starch solution obtained in experiment 
3 in a tube and add a drop of iodine so,ution. A deep blue color 
results. Now heat the ontents of the tube, the color disappears, 
to reappear on cooling. The blue dolor is due to the so called 
starch iodine, which possesses a variable composition. 
5) . To some of the starch solution add excess of tannic acid. 
A yellowish white precipitate forms or the liquid becomes highly 
opaque • 
6) Boil some of the starch solution with Pehling’s solution. 
No reaction takes place. 
7) Te about 50ec of the starch solution in a beaker add l/2cc 
©f H2SO4, cover with a watch-glass and boil for 15 minutes. 
Replace the water that may be lost by evaporation. pow place some 
©f the liquid in a tube, render alkaline with sodium or potassium 
hydrate, add some FehlingTs solution and boil. If no reduction 
place, continue heating the ontents of the beaker for another 
15 mimutes and test as before. Inversion has takes place; a 
reducing sugar (glucose) has been formed, as in the similar exper- 
iment with cane-sugar* This experiment is the basis of the com- 
riercial manufacture of glucose. 
DEXTRINE, c6H10°5- 
As explained above, a number of compounds are included under 
this head. They are the first hydration produets ©f starch. 
The commercial dextrin// is prepared by heating starch to 150-160° 
with ©r y/ithout water; also by drying starch at 100° previously 
suspended in very dilute nitric acid; or by treatment with a/tlds or CARBOHYDRATES 
16a 
malt and subsequent precipitation with alcohol. The behavior ©f 
the several *. . varieties ©f dextrine to iodine has been indi- 
cated abeve axid will be demonstrated in connection with the work • 
©n s&liva. 
Unlike starch, dextrine is very readily soluble in water. 
The solution is not fermented by yeast, but must first be hydrated 
further to maltose. Test a 1/f solution as follows: 
1) T© some of the solution add tannic acid—No precipitate; 
distinction from starch, gelatin, albumin. 
2) t© some of the solution add a drop or two of iodine solution. 
What is the result? And to what is it due? 
3) <p© some ©f the s©,ution add Fehling's solution and boil. 
Ordinary dextrine contains, more or less of reducing substances. 
GLYCOGEN, (C6H10Og)n. 
This curb©hydrate was first discovered in the liver and has 
since been shown to be present, in greater or less amount, in all 
the tissues of the animal body. q?he amount of glycogen in the liver 
will vary according to the food. Ordibarily it constitutes 
from l-4/o but may, after a rieh carbohydrate diet, amount to 12-16$• 
In fresh musele it amounts t© about 0, 6$ and disappears from the 
muscle as a result of work or starvation. It is pre-sent in Liebig's 
meat extract Although present in small amounts in nermal 
bleed, it is considersvlv increased after extirpation of the pan- 
creas. It is present in larger amount in pus, and yin leucocytes. 
Undoubtly it is a constituent of all living animal *' • cells. 
It is abundant in embryonic tissue and the liver of a j.w»-b©rn 
dog has been found to cg ntain as much as It is present in 
considerable quantity in melluses, notably in oysters. It has been 
found in sertain plants, notably fungi, as truggles; also in mueor 
and in the yeast, 
Glycogen is related to dextrine and to amylodextrin or soluble 
starch. It has the same precentage composition as starch or dex- 
trine. rphe exact formulae is not known. It would seem that the 
multiple 1 n* in the above formula is 6 although some place it at 10. 
Glyeegen is derived form the food, mere especially the carbohydrates. 
The excess of carbohydrates, whether starch or various sugars, 
is promptly stored up in the liver to be given off under the in- 
fluence of ferments according to the need of the body. Exclusively 
proteid diet likewise gives rise to glycogen. 
Various diastatie ferments such as are present in malt, saliva, 
panesreas, blood, liver, etc. invert glycogen. change is sim- 
ilar t© that which starch er soluble starch undergoes underlike 
ecnd.itions. mhat is to say, Erthyro dextrines, achroodextrine, 
iso-maltose, maltose, and eventually glueose form. On heating with 
water at high temperature, cr with dilute aeids, a similar hydration 
results. Owing to the action of these ferments ©f the body 
it fellows that in dead liver or muscle the amount of glycogen 
rapidly decreases, and is replaced by a dextrine body, ©r bymaltose 
©r glueese. CAHBOHYBUf B8 
16b 
The following table shows the relation of glyeogen to sugar 
In a 2rabbit; liver at different periods after death. • 
10 minutes* : 24 hsurs• • : 48 h©urs« : 
» 
! • • « • * 
• • • • • • 
Sugar : fflyesgen: Sugar : Glyoogeji: Sugar :Glyeogsn : 
0.75/ : 9.56/ : 5.58/ : 6.35/ : 3.85/ : 4.28/ : 
Tha action of the diastatie ferments is most marked in neutral 
or very slightly acid solutions. A 1% solution of sodium carbo- 
nate inhibits the change and so docs the acid solution ©f CO^. 
It is possible that the earbonie acid prevents or retards the hy- 
dration of glycogen in the body. glycogen is net affected 
by yeast. *>’*. au 
Glycogen is ah ’’amorphous, white, tasteless powder which dise 
solves in warm watew-'to form an opalescent liquid. The opales- 
cence disappears on the addition of an acid or alkali. On the • 
addition of Iodine the Solution beefefaes red or brown (Erythro- 
dextrine). u The eoler like that of starch iodine diaappears on 
heating. solutions are strongly dextro-rotatory. It is pre- 
cipitated from impure solution by alcohol. 
l). Isolation ©f Glycogen.—The following method gives the best 
results. It may be applied to 50g ©f perfectly fresh liver, or 
to l/s pint of oysters. The material is cut up as fine as pos- 
sible. If liver is used it pc put through a sausage machine• 
Tq the material then add 10 parts ©f boiling water slightly acid- 
ulated with acetic acid. Strain the opalescent liquid through 
muslin. £h$s liquid contains besides glycogen seme proteids and 
gelatin* T® remove the latter first concentrate t© a small vol- 
ume, then add alternately a few drops ©f HC1 and of potassium 
mercuric iodide till a precipitate ©eases to form. Finally £LIter 
off a little ©f the liquid and test it with acid and reagent to 
make sure that all the preteids are precipitated. If this is the 
case strain the liquid through muslin, then filter through paper 
and to the filtrate add two volumes of alcohol and. stir thoroughly. 
Allow the glycogen t© settle, then filter aff, wash with dilute 
alcohol (2 parts alcohol to 1 part water). Finally transfer to 
a beaker, cover with absolute alcohol and let stand an hour ©r more# 
Then filter off the glyc©gen fold the filter and gently squeeze 
off excess of alcohol, finally press between several layers cf 
filterpaper till dry. Pev/der, if necessary. 
The reagent employed above is prepared by mercuric iodide 
to a w&rmed solution ©f KI till it ceases to dissolve. The 
liquid is then cooled and filtered. 
With glycogen isolated as above make the following tests# 
1) T© seme glycogen in a small beaker add 20~3Gec ©f water 
and warm. The glycogen dissolves forming an opalescent liquid# 
Resemblance to soluble starch. 
2) To a portion ®f the solution just obtained add U few &rop.s 
Iodine solution (in potassium iodine). A reddish brown color 
forms. Then heat the contents of the tube. «?he color disappears 
to reappear on cooling. Resemblance to Erythrede&tria and t® 
starch itidine. The presence of pepton interfere# 
3) Boil another portion ©f the glyoogem. s©2u&i«n with Pehlingl€ 
*$©lALti©n. Note the r&suit. CARBOHYDRATES 
16c 
4) To seme ©f the glycogen solution add a few drops of HC1 
and boil a few minutes. Then co©l and neutralize, and test a por- 
tion with iodine; another portion with Fehling’s solution. 
Eomparc with Exp. 2 and 3. 
5) To some of the glyeogen solution add about l«c of saliva 
and mix. At the end of 10 minutes examine a portion with iodine; 
another portion with Fehling's solution. What is the result? 
CELLULOSE, (6H1Q0&)n. 
Cellulose, er weed-fiber, is present in all higher plants 
and. as a rule in the lower plants including fungi and bacteria. 
It largely makes up the walls ©f the cell. Cellulose is probably 
formed by the protoplasm of the cell out of the carbohydrates 
that result from the assimilation ©f the carbonic acid of the air. 
The molecule of cellulose is probably much more complex thah that 
of starch. Moreover it is probably that there are various dis- 
tinct cellulose bodies. Tunicin or animal cellulose is found in 
seme lower animals as the Tunieata, and is identical v/ith plant 
cellulose and yields ©n decomposition dextrose. Cellulose has 
been reported in the lungs, blood and pus of tuberculous patients 
(Freund). 
Cellulose is characterized by its difficult solubility. 
It is insoluble in water, alcshol, dilute acids or alkalis. 
It is soluble in an ammoniacal solution of copper oxide or Schwei- 
zer’s reagent and from this solution it can be precipitated, un- 
altered, in an amorphous form by acids, alcohol or water. Cellulose 
is furthermore characterized by its reaction with iodine and con- 
centrated sulphuric acid. Treated with concentrated sulphuric • 
acid and with iodine it gives a blue color. This is due to a so 
called amyloid substance which , however, is not identical with 
the amyloid found in the animal body. Indeed tha latter is not 
a carbohydrate but probably a proteid. In place of H2S04 zinc 
chloride can be used. 
It does not melt on heating but turns brown and eventually 
decomposes yielding various products.some of which have consider- 
able industrial importance . Thus, there is formed methyl alcohol 
(wood-spirit), acetic acid, (wood-vinegar) and creasote (wood-tar). 
Concentrated sulphuric acid dissolves cellulose and if this 
solution is treated at once with water a gelatious precipitate of 
soluble cellulose or amyloid forms. If the acid is allowed to act 
longer, or the solution is heated, no precipitation takes place 
on dilution. Y/hen paper is rapidly immersed in concentrated 
sulphuric acid to which 1/4 its volume of water has been added, 
and when washed in water, amyloid which is first formed is preci- 
pitated on the paper. The result is the tough parchment paper. 
When the solution of cellulose in sulphuric aotd is allowed 
to stand for some time, then diluted with water and boiled glucose 
forms. Some kinds of cellulose yield mannose. Unlike starch 
boiling with dilute H2SO4 has but little affect. 
With concentrated nitric acid, or a mixture of nitric and sul« 
phuric (1-3) acids, it forms various so called nitro-celluloses, 
These compounds rade use of In several important preparation#, CARBOHYDRATE 8 
16d 
Thus> CollodlUM, Vfhich is used in surgery and in photography> ' 
is a mlxtu’r'o*"b'f tri- and tetra-nitro cellulose dissolved id fothfir* 
Gun-cottoft or pyroxylin is a mixture of the tetra- and he#n*n;ur&fci* 
Smokelee6 powder, which has revolutionized modern war-fare* 
may h© pttre gun-cotton, or gun-cotton mixed with nitrate of 
and potaesium, or gun-cctton mixed with nitro-glycerin in different 
proportions (TTobelite, cordite explosive gelatin). Powders are 
also made out of nitro-phenol (picric acid) and out of nitro-napb* 
thalers* 
A mixture of nitro-cellulose and cellulose can be drawn out 
ifttO long glistening threads resembling The cel* 
ItilOse which is the basis of ordinary paper is obtained from wood 
by heating with calcium sulphate under pressure. 
Cellulose has been obtained in the shape of 
for minute needles. Cotton and linen threads and Swedish filter 
paper ane practically pure cellulose. In the dry condition it 
is permanent but in the presence of water it readily Undergoes 
under the influence of bacteria, fermentative decomposition giv- 
ing rise to marsh gas. This bacterial decomposition takes place 
in the intestines and marsh gas, acetis and butyric acids are formed* 
The cellulose of the food increases the peristaltic action of the 
intestines and consequently considerable nitrogen may escape 
absorption. 
1) Examine under the microscope and sketch, cotton, linen, 
silk and wool fibers also hair. The linen fibers are a hollow tube 
v/ith a thick wall and hence retain their shape whereas the cotton 
fibers have thin T/alls which readily collapse and- produce the twisted 
character. 
2) Tear up a little "washed” filter paper into small shreds 
(or use cotton) and warm with fresh Schwiezer's reagent. The cellulO 
se dissolves. Adidulate the solution with acetic acid when it 
precipitates in an amorphous form. The Schweizer reagent is ob- 
tained by adding sodium hydrate to a solution of copper sulphate 
in the presence of NH4CI. The hydrate precipitate is 
off, washed and dissolved in 20/f ammonium hydrate. 
3) Immerse some shreds of "Washed" filter paper, or cotton* 
in a strong solution of potassium hydrate (1-1). Allow the re-* 
agent to act for 10-15 minutes till the paper becomes gummy. Theft 
transfer to a dish of water, and wash thoroughly, then acidulate 
with a little dilute hydrochloric acid and add scan© iodine solution* 
A blue color, due to amyloid, results. 
4) To some cotton or shreds of paper add 5-10 cc of ©old 
Sulphuric acid. As soon as solution results take a portion Of it 
OOol and dilute with water. A gummy precipitate of amyloid forms* 
Add iodine solution, it colors blue 
Allow the remainder of the acid solution to theft Stftftd for some 
time then dilute with water and boil for l/2 hourj Oodli neutralise 
with potassium hydrate and test with Fehling*s solution ffr sugar# 
$) * pi lute some sulphuric acid with its volume Of 
water and cool the mixt'vfe. Then immerse* for a fet? seconds* aft Or* 
dinary filter paper; remove at once and wash iu t The 
tough parchment*papor results.- 17 
CHAPTER III. 
PROTEIDS. 
4. EGG albumin. 
Apply the following tests which are, more Or less, general re- 
actions for proteids to a 2% solution, unless otherwise indicated, 
of egg albumin. A white of an egg is carefully poured into an evap- 
orating dish, then cut uc with scissors and 20cc of the liquid is di- 
luted to 1 liter 1-50 (2%). After thorough shaking in a cylinder 
the liquid is filtered and the clear filtrate employed for the tests. 
Observe the frothing of the liquid on shaking. 
Dilute 2cc of the egg albumin to 10cc and shake thoroughly 
(1-5) 20%\ also, dilute 2cc to 4cc and shake till thoroughly raized 
(1-10) lQjS. 
COLOR REACTIONS OF PROTEIDS. 
The following color tests (1-6) are general reactions for pro- 
teids. 
1) Biuret test.—To the albumin solution (1-50) add an equal 
volume of strong sodium or potassium hydrate. Then heat to boil- 
ing and add 1-2 drops of very dilute CuSO^solution. The solution 
becomes colored, pink to violet, according to the amount of copper 
sulphate used. An excess of copper must be avoided. Salts of 
nickel give a similar reaction. 
Repeat the test omitting the heat. What is the result? 
All proteids give the biuret test, some more readily than Others. 
The hydrated proteids, albumoses and peptones, give the test in the 
cold, Gelatin gives in the cold, a bluish violet color, not purple 
red as in the case of peptons. 
The biured reaction would indicate that proteids contain the 
biuret or urea group. Diamids, such as oxamid and its derivatives, 
however, give similar biuret reactions, and it is possible that 
such diamid groups are present in the proteid molecule. It is pos- 
sible to remove the diamid group and the proteid that results no 
longer give the biuret reaction (Schiff), 
2) , Union's react ion.--To some of the albumin solution 
add a few drops of Millon’s reagent. A white precipitate forms 
which on boiling for 2-3 minutes becomes colored red- The liquid 
may become likewise red. 
This reaction is due to the aromatic nucleus contained in the 
proteid molecule. It is given by phenol, tyrosin, etc. 
Millon's reagent is prepared by dissolving in the cold 1 part 
of mercury in 1 part by weight of concentrated HN0I3, (1.40). Oentlj? 
heat is finally applied and when all is dissolved 2 volumes of water PROTEIDS* 
18 
are added. The mixture is allowed to stand for some hours and the 
clear liquid is then decanted from any crystalline sediment that 
may be present. 
5). Xanthoproteic reactions.--To some of the altaumin solution 
(1-50) add^an equal volume of cone. HN03 Then heat to boiling till 
the precipitate turns yellow or gives a yellow solution. Cool and 
add an excess of or NaOH. The color changes to an orange 
yellow. 
This test can be always incidentally applied to the precipitate 
Or liquid obtained in Heller’s test, or in the nitric acid and heat 
test (1, ). 
4) « Adamkiewicz’s reaction.--To 2cc of concentrated Ii£S0 add 
about 4cc (2volmmes) of glacial acetic acid and mix. To the4mix~ 
ture add 1 drop of dilute egg albumin. The liquid changes, slowly 
on standing, more rapidly when slightly warmed to a beautiful reddish 
violet color. The reaction is not given by gelatin or gelatin 
pepton. 
The presence of water interfere with the reaction. It is there- 
fore desirable to use the dry proteid or 1 drop of a concentrated 
solution. The spectrum of the solution resembles that of urobilin* 
5) Liebermann’s reaction.—To about 3cc of cone. HG1 add .1-2 
drops of undiluted egg albumin. Boil the liquid for several 
minutes. A pink to a violet color develpps. Too much water in~ 
terfers with the reaction. 
6) Heat some albumin with cone. H^SQ^and a little sugar* A 
red color results. Excess of sugar interfere by imparting a dark 
caromel color to the liquid. 
The proteid molecule contains one or more aromatic groups. 
This is seen in the fact, that on decomposition three distinct groups 
of aromatic bodies # . /form. This we may have 1st-)- the oxy-phengl 
group represented in phenol and in tyrosin: 2nd)- the phenyl group 
represented in phenylacetic acid; and 3rd! the indol group repre- 
sented by indol and skatol. The Xanthoproteic reaction is due to 
the formation of intro-products and is also due to the presence of 
the 1st group. Millon’s reaction is due to the presence of the 1st 
group of compounds. It is not given by the 3nd or 3rdgroups. 
The Adamkiewicz reaction is due to the 3rd group of products. 
On the other hand the Liebermann’s reaction is apparently not due 
to the aromatic group. 
PRECIPITATION REACTIONS OP PROTEIDS. 
7) Take 4 test-tubes label and equip as follows: To tube 1 add 
l~2cc of the undiluted egg albumin; to tube 2 add 5cc of the egg 
albumin, 1-5J t.n tub* a *** FHOTHIDS . 
19 
and» to tube 4 add See of the solution, 1-50. Immerse the 4 tubes 
in a boiling water-bath for 5-10 minutes, after which examine and 
note the resuite. Test the reaction of tubes 2,3,4. Tube 1 coag- 
ulates solid, whereas tubes 2,3,4 are more or less opalescent 
but far from coagulation. Dilution of egg albumin with water 
renders it non-coagulable by heat. Compare with test, Blood-serum 
IV, 4. 
6). In each of 4 test-tubes place 5cc of the egg albumin sola-- 
tion (1-50) . To tubes 1 and 2 udd respectively lec and 0.2cc of ft 
10)4 NaCl solution. To the tubes 3 and 4 add respectively 1 and 5 
drops of a 1% acetic acid solution (lec of glacial acetic diluted 
to lOOcc) To a fifth tube containing 5cc of egg albumin solu- 
tion (1-10) add lec of a 10/if NaCl solution. Immerse the 5 tubes 
in a boiling wator-bath for about 5 minutes, then examine and note 
the results. Test the reactions of tubes 3 and 4. In experiment 
7, above, tube 4, which can be considered as a control for this eXpcr-4 
iment, on exposure to 100° shows only a very slight opalescence. 
The addition of a small amount of NaCl increases the opalescence 
(tube2); the same amount of NaCl as in tube 1, added to a stronger 
solution of albumin (tube 5) brings on coagulation on heating; 
and a larger amount brings on partial coagulation on the walls of 
the tube (tube l). Now add 1 or 2 drops of the 1% acetic acid 
to tubes 1, 2, 5 and to tube 4 add lec of NaCl and heat again. 
Prompt and complete coagulation results. The liquid is clear. In 
tube 3 the addition cf one drop of the diluted acid, thus changing 
the liquid to a very slight acid reaction, suffices to produce on 
heating a precipitate. A very slight excess of the acid (as in tube 
4) prevents coagulation by heat. If NaCl however is added coagu- 
lation promptly results. 
In attempting to remove albumin completely from a solution, a 3 
in the case of urine, it should be remembered that very dilute sold*" 
tions must be barely acidulated with acetic acid. Furthermore, that 
the presence of NaCl ’favors coagulation on subsequent heating. 
Albumin coagulates in a slightly acid or neutral solution, 
especially in the presence of a neutral salt or ./NaCl. Globulin 
requires a neutral salt to keep it in solution ana this moreover 
favors coagulation on heating. Haemoglobin on heating decomposes 
into haematin and globin; the latter as just stated coagulates 
on heating in the presence of a neutral salt. Neucloalbumin is 
coagulated or thrown cut of solution by acetic acid alone. The 
albumoses as will be seen later are precipitated by NaCl and the 
precipitate unline albumin and globulin dissolves on heating. 
Peptons are not coagulated by heat 
9).To about See of the albumin solution (1-50) add an e :ual 
volume of concentrated HNO3 so that the two liquids do not mix. 
This is done by allowing the acid to slowly run down tjhe dide of thO 
inclined tube. A white cloud forms at the zone of contact of the 
two layers (Heller’s test). Now mix the two liquids and gently 
warm. A fluoculent precipitate separates. Now heat the mixture 
to boiling. In a short time the precipitate dissolves, acid albumin PROTEIDS 
20 
forms, and the liquid is colored yellow. Cool the liquid and add 
an excess of NH4OH. An orange yellow color results(Xanthoproteic 
reaction). 
Egg albumin is therefore coagulated by HNO3. The solution of 
this precipitate on boiling shows a distinction between this and 
the serum proteids. 
, Mineral acids, such as HNO3, coagulate albumin and globulin. 
The albumoses are precipitated by HNOj especially if NaCl is present, 
but the precipitate readily dissolves on the application of heat 
and reappears on cooling. Peptons are not precipitated by acids. 
a). The test employed most often for the detection of albumin 
(and globulin) in the filtered urine is the coagulation or nitric 
acid and heat test. The reaction when properly carried out is ex- 
ceed irgTy deTicate. The best proceedure is as follows: To the 
urine add some concentrated HNO3SO as to form two layers (see above). 
A precipitate or cloud indicates albumin. Now mix the two liquids 
and heat. A presistent flocculent precipitate is due to the al- 
bumin or globulin or both. Should it be necessary to decide whether 
this precipitate is due in part or whole to albumins, it can be done . 
by saturating the urine with MgSCUaccording to directions given 
underftlobulin Test 5. 
If heat is applied direct to the urine a precipitate of 
phosphates may form. This, however, dissolves readily in HNO3. 
If the urine is alkaline the HNO3 should be added first to prevent 
formation of alkaline albuminate. 
Apply the test as just given to some albuminous urine. 
10) . To about 5cc of the albumin solution (1-50) add 1-2 drops 
of strong acetic acid, then add 1-2 drops of potassium ferrocyanide* 
A voluminous precipitate forms. 
This is a very delicate test for all proteids. It is not 
given, however, by peptons. The presence of NaCl favors the pre- 
cipitation of the album oses. Moreover the albumdme precipitate 
dissolves on heating and reappears on cooling. 
This test and the nitric acid heat test, given above, are com- 
monly employed for the detection of albumin in the urine. 
If, in the of urine, the amount of the precipitate is 
small and its nature doubtful it should be transferred to a filter 
and washed. The precipitate can be transferred by means of a glass 
rod to a test-tube and Million’s reagent added, If on heating a 
reddish coloration gofrns it indicates &he presence of a proteid. 
Another procedure is to add l/2cc of the boiling Millon’s reagent 
direct to the precipitate on the filter(Winternitz). 
11) Strongly acidulate some of tho albumin solution (1-50) PROTEIDS 
21 
;73r 1 T‘ul w’;: a fcv; of phosphotungstic acid. A heavy 
vv*^-■ precipitate results. It is given by all proteids. Phospho- 
nclyldic acicl behaves in a similar manner* 
12) Acidulate another portion as above and add a few drops 
of a solution of potassium mercuric iodide. ::ote the results. 
Vhy was "his reagent used in the preparation Of glycogen? 
15) a portion of the albrain solution (1-50) add 1-2 drops 
of tannic acid. That is the behavior of tannic acid to starch? 
To dextrin? 
ii). To another portion of the solution add a few drops of 
picric acid. A yellow voluminous precipitate forms. This reagent 
is used in Esbach* s method for the estimation of albumin in urine. 
The r- .yds employed in tests 10-14 i: clue ice are some :.ifces 
r. pc hen of r-.s alhaloidal reagents because of their reactions with 
the alkaloids and other bases. They are general reagents 
for proto ids. 
] ). To hoc of the albumin solution (1-50) add one drop of 
mercuric chlorid. A heavy white cloud or precipitate results. 
Divide the cloudy liquid into two portions. 
a) To ere add an equal volume of a 10$ solution cf NaCl. The 
precipitate promptly dissolves even if mercury is in large excess. 
b) . To the other portion add two volumes of the diluted egg 
solution and nix. The precipitate dissolves if too much mercury 
has not been added. 
16) To another email portion of the egg albumin solution add 
1-2 drops of dilute lead acetate *n/i note the result. 
17) a To a portion of the solution add 1-2 drops of silver 
nitrate,. A voluminous white precipitate forms which on the addi- 
tion of 1THr.OH dissolves. 
Experiments 4, 5, 6 are made with the salts of the heavy rcet.als 
which precipitate most of the proteids. Why is the white ol bggs 
administered in case of poisoning with corrosive sublimate or with 
salts of ether heavy metals? Why should a stomach pump be sub- 
sequently used? 
13) To about 3cc of the albumin solution (1-50) add 10~15cc 
of strong alcohol and mix. If no precipitate forms, but merely a 
cloudiness, than add l/4~l/2ec Cf ft \Q% solution of NaOl. A volum- 
vfcpc tltfrttffc* fwMMur* 22 
Alcohol added in large excess (10 volumes or more) precipitate# 
all proteids. The presence of NaOl favors the precipitation. 
19) Place lOcc of egg albumin (1-50) solution in a small beaket* 
at* test-tube or pot. Add about 7g of powdered and immerse 
in a water-bath at about 35° for half an hour, St infrequently 
till the salt ceases to dissolve* Notice the heavy white precipitate 
that forms (albumin and globulin)# When saturated transfer the 
contents to a dry filter. Test the filtrate: 
a) By acetic acid and heat. 
b) By the biuret test in the cold. 
20) Place lOcc of the egg albumin solution (1-50) in a small 
beaker, as above, add about 12g of MgS04 and digest, with frequent 
stirring, at 35° for about half an hour. Observe that only a 
very slight cloud or precipitate forms (globulin). Filter through 
a dry filter and test the filtrate as in Exp. 19. What proteid 
is frequent in the filtrate? In the biuret test a large excess 
of NaOH should be added owing to the precipitate of Mg(0H)2 that forms. 
21) Determination of the coagulation point of albumin.--Place 
about 5cc of the undiluted agg albumin in a test-tube. Close the 
tube with a stopper through which passes a thermometer. The bulb 
of the thermometer should hearly touch the bottom of the tube and 
should be completely immersed in the albumin. Suspend the tube 
thus equipped in a large beaker of water. Fully two-thirds of the 
tube should be immersed. Heat gradually the water in the beaked 
and stir continnually by means of a glass rod bent at right angles* 
Note the temperature at which the albumin clouds. The alblifnin 
then becomes sticky : does not flow readily when inclined and finally 
becomes solid. Note the coagulating point of egg albumin. 
22) .To 20cc of the 2% albumin solution add 2-3 drops of 
concentrated HC1 and boil. No precipitate forms owing to the for- 
mation of an acid albumin.Cool the solution, a) to a portion add 
an excess of concentrated HC1 a precipitate formsthat is difficultly 
soluble in excess. b)then in the remainder that is good place a 
litmus paper and add-, drop by drop, very dilute NaOH. Mix the con- 
tents well after each addition of alkali. As soon as a precipitate 
or cloud forms note the reaction of the liquid. The precipitate 
of albuminate forms while the liquid is still acid. After the , 
precipitate has formed add 2-3 drops more of the dilute NaOH. 
It dissolves at once to form an alkali albuminate. 
23) To lOcc of the albumin solution add 1-2 drops of NaOH 
solution and warm gently for a few minutes. An alkali albuminate«■ 
forms. Raise the solution to boiling* It does not coagulated 
Cool, add litmus paper and carefully neutralise, as above, with 
dilute HC1. liThat is the result? What is the effect of a slight 
excess of HC1? PROTEINS 
23 
Report the results obtained with egg albumin and with proteids 
Subsequently to be studied in a tabular form such as the following! 
Egg peraihSerumAlbu rl 
’ept [ 
tela 
albu-JalbuHglobujaose .|t 
;on. It 
;in. 
min. *n 
lin. ilin. , 
Biuret 1. 
Milion 2- 
Xanthoproteic 3. 
Adamkiewicz 4. 
* : 
Boiling 
Nitric acid 9. 
Acetic and £srrocyanidelO 
Phosphotungsiic acid 11. 
Pot, Mercuric iddide 12 
Tannic acid 13 
Picric acid 14 
Mercuric chloride 15. 
I,e ad acetate 16. 
Silver nitrate 17, 
Alcohol 18. 
Ammonium sulphate 19. 
Magnesium sulphate 20. 
CHAPTER II. 
£ERUM ALBUMIN SERUM GLOBULIN. 
Globulin*—is usually associated with albdmin, though it may 
Sometimes, as in the urine, occur alone. The tests given for al- 
bumin, as well a1 the general proteid reactions, are also given by 
globulin* For the separate recognition of albumin and globulin, 
when both are present in solution, it is necessary to resort to 
precipitation by either of the following methods: 
1). PrecipitationRwith MgS04.—To lOcc of blood-serum, in a 
small beaksc or test-tube or pot, add lOcc of saturated MgS04 and 
15g of powdered MgS04. Immerse the beaker or tube in a water-bath 
at a temperature of 30-35*. Stir frequently for 1/2-1 hour, until 
the MgS04 ceases to dissolve and the liquid is saturated. The 
globulin is thrown out of solution. Transfer the liquid and pre- 
cipitate to a small filter. Save the filtrate (a) which contains 
albumin. When the liquid has drained, through .wash the raaldne 2-3 PROTEINS 
24 
times with saturated MgSO4. Finally spread out the filter on a 
flat surface, transfer the percipitate by means of a spatula to about 
20cc of water. Slobulin when pure does net dissolve in water 
but in this case, owing to the {.resence of salts, it dissolves*- 
Filter the solution and the clear filtrate (b) containing the 
globulin is reserved for experiment 3. 
to 
origional filtrate (a) which contains serum albumin 
the tents enumerated in the table. Note the results. Wherein 
does egr albumin differ from serum albumin? Boil £ portion of the 
serum albumin solution to coagulate the albumin. filter and apply 
the biuret test to the. filtrate. What are the results? 
2) Precipitation by semi-saturation with (NH4)0SO4.—To lOcc 
of blood serum as above, add 10 cc of saturated Immerse 
in a water-bath at 30-35° for about 1/2 hour and stir frequently. 
Then transfer the contents to a small filter. Save the filtrate <AJ 
which contains album. V;ash the residue on the filter 2-3 times 
with semi-saturated (NK4)3SO4* Finally spread cut the filter on a 
flat surface, transfer the precipitate tc about 20cc of water* 
The globulin precipitate dissolves for the reasons given above under 1. 
Filter the solution and combine the clear filtrate (B) with 
the corresponding filter from experiment 1. The resulting solution 
is used for experiment 3. 
The precipitate obtained by this method is larger that that ob~v 
tained by the method. The liquid filters much easier. 
3) Separation of salts from globulin by dialysis.-—'Place thd* 
combined filtEafees (B) in a dialyzer and dialyzo against running" 
watero Every day remove a few drops of the liquid from the dialyza? 
with a pipe!* and add to some dilute BaCl2 solution. The dialysis \ 
should contdime till all the sulphates are completely removed. \ 
This may require 3-5 days. In warm weather to prevent decomposltlnn. 
it is well to add a few drops of thymol. 1 
Then the sulphates have dialyzed out the globulin is thrown / 
out of solution as a white granular precipitate. Now transfer 
the contents of the diaUyzer to a small beaker. Pour into the 
dialyzer about 20cc of a 2% solution of NaCI and gently agitato 
to dissolve any precipitated g&lbulin. Add this saline solution, 
to the contents of the beaker and stir till the globulin dissolves.. 
Finally allow the liquid to stand for a while then filter. >phe 
clear filtrate new contains pure globulin. Observe the frothing 
of the liquid on shaking. 
To this solution of globulin apply the tests enumerated in the 
table on page 23. Make careful redords of the results obtained* 
ihe presence of NaCI will interfer with the tents 16-17* 
Boil a portion of the globulin solution to coagulate the -globulin 
To the filtrate apply the biuret test. 25 
4) . To lOcc of blood-serum add an equal volume of saturated 
Then add Sg of powdered (NH4) gS04 and immerse in a 
water-bath at 30-35°, stirring frequently,"for about 1/2 hour. 
rnhe liquod becomes saturated with (NH ) SO and a precipitate forms. 
Finally transfer the filter. Notice u:-ne perfect clearness of 
the filtrate. 
Test a portion of the filtrate by boiling; another portion 
with tannic acid. 
To another portion bpply the biuret test in the cold. 
The absence of proteids in the filtrate demonstrates that al- 
bumin and globulin and completely precipitated on saturation with- 
(NH4.) S04. The absence of a biuret reaction indicates the absence 
of a peptron . 
5) Detection of globulin in the urine.—As indicated under 
albumin (Experiment 9 a, page 20) the ordinary tests for albumin 
are also given by globulin. In order to ascertain positively which 
of the two, or if both are present it is necessary to resort to 
the saturation method with For this purpose lOOcc of the 
urine can be taken and neutralized. 120g cf powdered MgS04 are 
then added and the liquid kept at 30-35°, with frequent stirring, 
till the to dissolve. If globulin is present a pre- 
cipitate will"form. This can be removed by filtration washed with 
saturated MgSO,solution, and finally dissolved in water (See page, 
globulin II. it) rrhis solution of the precipitate can now 
be tested. It coagulates on heating especially if slightly 
acidffied with acetic acid. It gives the nitric acid and heat 
tests, also the biuret reaction. 
The filtrate fDom the Mg304 precipitate contains albumin if 
any is present. The tests just given applied ti this filtrate, 
if positive, prove the presence of albumin. 
A L E U M 0 S E III. 
This compound or rather group of compounds can be readily pre- 
pared from White’s or Schuchardt’s commercial pepton since this con- 
sists largely of albumoses. Albumoses are precipitated by sat- 
uration with (NH.) SO or with MaCl in an acid solution. 
* iCy 4 
A 20/b solution of the commercial pepton is used. The powder 
readily dissolves especially if the liquid is warmed and thoroughly 
stirred. 
ll. Place some of the solution in a test—tube and heat to 
bolding. The liquid does not coagulate thus indicating the absence 
of adbumin and globulin. PROTE ID 
26 
2). Tq IOcc of the solution ado. iOcc of saturated 
solution ''nr1 abo*’* °g of powdered (NR,) Saturate the liquid 
in a water-bath at about 30-35° according to the directions given 
in experiment II, 4. Notice the sticky precipitate that adheres 
to the rod and to the sides of the beaker or tube* Since albumin 
and globulin are absent (Exp? 1.) the precipitate that forms 
consists of albumCses. Transfer the precip&Oate to a filter 
and wash with about IOcc of saturated (NH ) 30/. 
4 4 
Save the filtrate (A) for subsequent tests for pepton. 
By means of a glass rod gather the sticky albumose predipitate 
and transfer it to about 20cc of water in a test-tube. While 
stirring, heat the liquid carefully and the albumose dissolves com- 
pletely. 
With this aqueous solution of pure albumose make the following 
tests, employing small quantities of the liquid abcufe lcc* 
a. Heat a portion to boiling. It dees not coagulate# 
b. To a portion add NHO , drop by drop. A slight precipitate 
/may form which dissolves and gives a yellow solution. If there is 
no permanent precipitate add some saturated NaCl, drop by drop, 
till a precipitate does form. Now heat gently the contents of 
the tube. precipitate dissolves and on cooling reappears. 
This reaction is characteristic for the albumoses. 
In the absence of NaCl some of the albumoses, especially 
dentero albumose, do not give a precipitate with NHO3 An excess 
of NaCl, however, should be avoided since In that case the albumose 
precipitate does net dissolve completely on heating. 
c„) To a orticn of the solution add a few drops of acetic 
acid (1-10) and 2-3 drops of potassium ferrocynide. If no pre- 
cipitate forms add NaCl according to directions given above under c.); 
A precipitate then does form and on heating gently it dissolves. 
On cooling the solution it reappears. 
This reaction, like the predeeding, is also characteristic 
if albumoses. A certain amount of NaCl is necessary as in the 
NHOjj test. 
d.) To about lcc of the solution add 1-2 drops of dilute 
acetic acid and about 5cc of a saturated NaCl solution. A pre- 
cipitate or cloudiness results. On heating this disappears, 
to reappear on cooling. 
To the remainder of the solution of albumose apply the tests 
given in the table (page 23) and note the result. In which of 
these reactions will the presence of chlorids and of ammonium 
salts interfer? 
Apply the biuret test without the aid of heat. The hydra- 
tion proteids give this reaurtrion readily in the coldv PR0TEID3 
27 
3). Detection of albumose in the urine. 
The reactions given above under 2, especially b, c, 
and d, are characteristic Of albumoses. To detect albumos in the 
VTtne, or in other liquids* it is necessary first to remove the 
albumin and globulin. This can be readily done by acidhlpihnp: 
very little with acetic acid and applying heat. The albumin and 
globulin coagulate. To the filtrate the biutet test can be applied. 
If the result is negative it indicates the absence of albumoses 
and also of pepton. If, however, the result is positive it is due 
either to albumoses or to pepton, or to both. The tests given 
above under 2b, c, and d can now be applied and if positive* 
the presence of albumose is demonstrated. If these tests fail 
the positive biuret reaction is due to pepton. 
PEP?ON, IV. 
Pepton is not precipitated by (NH4)2S04. The filtrate (A) 
obtained in experiment 2 under albumose therefore contains pepton 
if it be present. 
To this filtrate apply the biuret test in the cold* A pos- 
itive reaction is due to pepton. As indicated before the hydrated 
proteids, as a rule, require heat, 
To obtain a pure solution of the pepton it would be necessary 
to resort to dialysis, or to treatment with baryta on a water-bath 
to remove the (NKjJgSO^. 
To the origional filtrate containing pepton apply the tests 
given in the table on page 23 and note the results. With which 
of these reactions will the (NHJ0SO4 present interfer? 
Detection of peptons. 
To about 5Q0cc of the urine, or to an aqueous extract of the 
tissue to be examined, made at about 40°, add just enough lead 
acetate to give a strong preeipitate and filter. This removes 
mucin* Test the filtrate for albumin and if present remove 
in the following manner: Add a little sodium acetate and then con- 
centrated ferric chloride till the mixture is blood red in color* 
Then neutralize with potassium hydrate (or leave slightly acid), 
boil, cool and filter. filtrate should give no precipitate 
with acetic acid and potassium ferrocyaaich (absence of iron and 
of albumin). If it is perfectly free from albumin make the folio* 
ing tests: 
1. Add acetic acid and phosphotungstic acid--a cloudiness 
forms on standing if pepton is present. 
2. If pepton is indicated by the above trial it can be is- 
olated by the following method: Add 0.1 volume of concentrated 
hydrochloric acid and then phosphotungstic acid also acidulate 
with hydrochloric acid, as long as a precipitate continues to 
form. Filter at once and wash with dilute sulphuric acid to 
5cc in lOOcc water), till the filtrate is colorless. While the pre- 
cipitate is still moist mix it with an excess of pov/dered barium 
hydrate, add a little water, gently warm for a short time and flit er 
To the filtrate which contains pepton apply the hiurefc test, 
(Hpfmeister7 s method). PROTEIDS 
28 
This method does not indicate true peptones only, but also 
albumoseD 
Another method for the detection of pepton is based upon its 
behavior to (NK)S04. The method as employed by Devote is as 
follows: To of the urine add 80% by weight of (NH4)2S04. 
This is added to urine even if albumin and globulin are absent 
in order to remove neucloalbumin. Warm the mixture on the water- 
bath till the salt dissolves, mhis will occur in 10-15 minutes,. 
how place the beaker in a steam sterilizer for 30-40 minutes or 
longer o The albumin coagulates cosap lately irrespective of the 
reaction of the fluid. The mixture is allowed to cool, then filtered 
The filtrate can be tested by the biuret reaction. If positive 
pepton is present. It can further be precipitated with'tannic 
acidc 
The residue on the filter can be washed with hot water will 
the filtrate ceases to give a test for BaSOx. If the filter has 
previously been dried and weighed, and is now again dried and weighed 
the difference is due to the albumin and globulin. (See estimation 
of albumin and globulin.) 
The first portions of the hot wash-water are collected! combined 
and tested by the biuret reaction. If positive it is ordinarily 
said to be due to pepton (Dcvoto, Jakash.) 
The Hofmsister method will often give positive results where 
Devoto:s method fails. 
In reality the reaction in that case is due to aibumoses* 
The true pepton which would be present in the filtrate from the 
cold saturated solution seems to be very rare in mine. 
As used in a clinical way the term "pepton" includes pepton 
and aibumoses, Such pepton may be present, though not always9 
in the blood of the leukaemj.es dufcing life. The blood obtained 
from deceased leukaemics, especially if decomposition has set in 
is rich in such pepton. The normal liveSr does not contain pepton. 
whereas the spleen dees. The liver and spleen of leukaemics 
is rich in such pepton. 
Another process for the detection of true pepton is as followst 
Saturate the solution at the boiling point with ammonium 
sulphate and filter while boiling hot. Allow the filtrate to cool, 
decant the liquid from the crystals which separate, dilute strongly 
and precipitate the pepton by cautious addition of tannic acid, 
pee stand for 24 hours then filter. Boil the precipitate for a 
few' minutes with baryta water filter and from the .filtrate remove 
the excess cf barium by passing carbonic acid. Iilter off the 
barium carbonate and test the filtrate for biufcet. Hoteids 
29 
GELATIN, V. 
To study the reactions Of gelatin a 2% soMtion of the best 
French gelatin (silver) is employed. 
1) . Shake up some of the solution. Notice the foaming of 
the liquid. 
2) „ To a portion of the solution add some bromine water. 
An abundant3 yellow, sticky precipitate forms. 
3) In each of two test-tubes add 1-2 drops of saturated HgClg 
soluyion. To tube 1 add about 5cc of the gelatin solution. To 
tube 2 add an equal volume of water. Then add to each tube some 
HgS-water and heat. Tube one is dark yellow but contains no pre- 
cipitate, whereas tube 2 has a blackish precipitate of HgS and the 
liquid is clear. Gelatin prevents the precipitation of mayy 
Otherwise insoluble, compounds. 
4) To the gelatin solution apply the several tests given 
in the table on page 23 „ Tabulate the results, and carefully 
note the differences. 
Observe that the heavy met aid do not precipitate gelatin* 
whereas the other proteids are precipitated. Also, that gelatin 
is not precipitated by ferrocvanide even in the presence of NaCl 
and in this respect it resembles pepton. 
The Xanthoproteid reaction is weak owing to the absence of 
the phenol group, C5H5OH,, The biuret reaction applied to the Cold 
solution cf gelatin gives a bluish violet color, whereas; pepton 
gives a purple Millon's reagent gives a white preci- 
pitate thich on heating becomes red and the liquid becomes pink* 
It is probably that &&1 the other reactions are not strictly due 
to the gelatin but to a mixture of some pepton or albumose. CHAPTER IV. 
SALIVA. 
baliva is a mixture of the secretions, of the parotid* submaxAl** 
lary? and sublingual glands.*• The reaction of mixed saliva is usualjty 
alkaline > but may D?l Tasting also during the light toward morning, 
and after meals, or after much talking, become acid* It 
also becomes acid on standing a few hours, (Repin1* «. It is more or 
less opalescent and viscid and foams readily, Tire character if the 
saliva will vary according to which gland furnisnss the most of the 
secretion. The prot&id gland yields a fluid secretion whereas the 
submaxillary and lingual glands yield slimv secretions. In 
°ebrile diseases the secretion of sa'liva may be diminished or wholxy 
suppressed, and hence dryness of the .south and throat, as well as 
altered taste, A decrease is also observed in diabbees, in severe 
diarrhoeas, as in cholera. The adm .filtration of potassium iodide 
or of mercury produces an increased flow, or salivation, and the com- 
position of the saliva itself becomes altered* Albumin becomes 
present and the amount of salts in solution is increased. An in*-* 
creased flow of saliva (ptyalism) is also brought about by irritant 
poisons such as acids and alkalis; also by certain foods, lemon? etc*, 
and occurs also in some diseases, especially in inflammatory condi- 
tions of the mouth, tonsil, and palate* 
In icteric conditions the saliva does not contain bile constitu- 
ents* In diabetes it does not contain sugar. In the latter case, 
however, the action may be acid because of lactic acid. In nephritis, 
urea may be present in the saliva, and uric acid has been found in 
uraenic conditions, Leucin has been found in the saliva of a hys- 
teric case. 
Salivary calculi which occasionally deposited in the sali- 
vary ducts consist chiefly of calcium carbonate and phosphate with- 
organic matter. The tartar deposited on teeth has essentially the 
same composition,, the phosphates however predominate. These calci- 
um salts are held in solution in the saliva by carbonic acid, J§n 
exposure to the air this passes off and the salts are deposited* 
The. specific gravity of the mixed saliva varies from 1,002 to 
1.003 and -ontains 1/2-1/f of solids which consist of albumin, mucin, * 
ptyaliO, traces of urea and other nitrogen compounds and mineral 
constituents, The amount of saliva secreted in the course of 24 
hours lb 1400-1500 cc, The flow is increased after meals and by 
pilocarpin. Atropin diminishes salivary secretion* 
The chemical examination of saliva has at present but little 
clinical•significance. Physiologically, however, the composition 
and action of saliva is of the greatest importance. The ferment 
or enzyme present in the saliva is known as ptyalin and possesses 
a d.tastatis or amylolytic action* That is, converts starch into 
then intn and maltose. Eventually glucose farms 
probably however tne result of the action of an inverting ferment*: 
jp-yaJAzl tff “hot present in the saliva of all animals. The parotid 
oS new-born contains ptyalin, whereas the submaxillary 
saliva does contain it for several months. In the saliva of 
some animals as “horse’ the ferment is not present free but as a 
zymogen from whiats It rasui-* 1 y forms in mastication. This as well 
as -he enzy* * -► irsi-gf-icd dnwn cschani ca-Tly by a. precipitate SALIVA. 
31 
ef calcium phosphate and this fact is utilized to obtain the ferment 
in a comparatively free state. 
Although ptyalin reserablesin its action the diastase of malt, 
it is different. This is seen in the fact thp.t the former acts best 
at 40°, the latter at 50-60°. The amount of ptyalin present in the 
saliva is subject to variation. HOI not only prevents the action 
but it also destroys the ferment. The action of the ptyalin is mst 
marked in neutralized or very faintly acid saliva. 
A microscopic examination of the saliva will always show epithe 
lial cells from the mouth and tongue, also salivary and mucous 
corpuscles. Bacteria are always numerous, and certain species as the 
leptothrix, spirillum, and spirochaete are almost invariably present. 
Among the pathogenic forms found in the mouth in health ar in disease 
my be mentioned the bacilli of diphtheria, tuberculosis and tetanus, 
Eraenkel*s diplococcus, the micrococcus tet rage nils and the pus-pro- 
ducing staphylococci and streptococci, the fungus of thrush and of 
avtive mycosis. Blood or pus cells may be present in the saliva 
in inflammatory suppurative conditions of the mouth, gums, etc. 
Rub the tongue thoroughly over the inside of the mouth, teeth 
and gums, collect the saliva and examine under the microscope for 
epithelial cells, salivary corpuscles, etc. 
The saliva necessary for the following experiments can be readily 
obtained by chewing a piece oT pure paraffin. * Commercial gum must 
not be used inasmuch as it contains sugar. Collect about 100 cc. 
of saliva. 
1) Test the reaction of the mixed salivawith litmus paper. What 
is it? 
2) Nearly fill a 50 cc. graduate with saliva. If there is 
any foarr on the surface remove it with a piece of filter paper. Then 
immerse an urinometer and note the specific gravity of mixed saliva. 
What is the reading if immersed in pure water? 
3) To about 5 cc. of saliva add a few drops of acetic acid (1-10) 
and gently agitate. A flocculent precipitate of mucous forms. 
4) To some saliva apply the biuret test(Exp. p. ). The 
result is due to mucin. 
5) To some saliva acid a drop of nitric acid and boil. Is al- 
bumin present in saliva? 
6) To the contents of the tube from the preceding experiment 
add KHzOH. An orange yellow solution forms xanthoproteic reaction. 
7J. To scme saliva add a few drops of Millon’s reagent. A 
heavy yellowish precipitate forms which on boiling becomes reddish. 
This is due ,o mucin. 
5). To some of the saliva add a drop of dilute HC1, then, drop 
by drop, dilute ferric chloride till a red coloration results* This 
is due to the formation of ferric sulphocyanide. The reaction is 
more distinct kf after the addition of HC1 the liquid is filtered 
and the ferric chloride is added to the filtrate. 
9) To another portion of saliva add a little iodic acid and suae 
starch solution. Iodine is liberated and colors the starch blue. 
This is due to a sulphocyanido* Explain the reaction. 
10) To some saliva add a few drops of dilute mix; then 
add a few drops of a colorless solution of potassium iodide and 
finally a few drops of starch solution. Iodine is liberated and 
dolors the starch blue. This is due to nitrous acid. Explain • 
11) To some saliva add. a drop--or- tvro- HCl,theJo drops SALIVA. 
32 
of a saturated sulphaniiic acid solution and mix. Now add a few 
drops of naphthylamine hydrochloride. A pink or red solution indi- 
cates the presence of nitrous acid. This test is employed in test- 
ing for nitrates in water analysis. 
12). Take a small dose of potassium iodide, rinse out the mouth 
thoroughly with water and test some of the saliva, at Snce for KT. 
This is done by adding to some of the saliva a little chlorine—water 
and then shaking with carbon bi-sulphide. A pink coloration of 
tee latter indicates iodine. Iodine should be absent from the salva 
after rinsing. After that collect a little of the salive every 10 
minutes and test for iodine as above. How soon does KT appear in 
the salivaafter being taken into the stomach? 
15o Separation of Mucin.-- Pour 10 cc. of the saliva slowly and 
with constant stirring, into 50 cc. of absolute1 alcohol. A fi- 
brinous light precipitate forms. Allow to settle over night in a 
covered beaker. Then filter, wash the precipitate on the filter 
twice with alcohol, then with ether. Spread out the filter to dry 
and finally with a spatula, remove the white chalky powder of mucin. 
a. To a little of the powdered mucin in a tube add some 
water. It swells up but does not pass into solution. Then add a 
drop or two of KOH when it dissolves forming a milky solution. To 
this solution now apply the biuret test. What is the result? 
b. Place the remainder of the power sLn a tube and add di- 
lute HC1 (1-3) and boil for some minutes. Transfer a porti®n to 
another tube, cool, render alkaline with KOH and boil with Pehling 
solution. The formation of red cuprous oxide indicates the presence 
•f a reducing substance. If this test is not given, boil again, 
assi the remaining original liquid and again test a portion as above. 
Mucin is a complex proteid substance and on decomposition, as 
aoove, it yields a reducing compound which, however, is not sugar. 
What other substances on heating with an acid yield reducing substances 
14). Action of Ptyalin.— Prepare a starch solution according to 
the directions given under starch. Into each of the eight tubes 
place about 3 cc. «f Pehling*s solution. Into each of other 3et 
of 8 tubes place 1-2 drops of dilute iodine solution. 
a. To 30 cc. of the salt solution in a graduate 6 drops 
of saliva are added at once and mixed thoroughly. Immediately 
after mixing pour 2-3 cc. of the mixture into a tube containing 
Pehling solution, and also into a tube containing the iodine. The 
latter colors deep blue—due to starch. Hoil the tube with Pehling 
solution. No react ion should take place—absence of su^ar. 
At intervals of two minutes apply the test with Pehling solu- 
tion and with iodine to the mixture in the manner just given. Tabu- 
late your results, noting the time when sugar appears in the mix- 
ture; when erythro-dextrin and achroo-dextrin appear. The time-of 
appearance of the latter is spoken of as the achromic point. Y.'hen 
this is reached boil some of the starch mixture-with Barfoed’s re- 
agent. What is the result? What does this indicate? 
At the conclasion of this test add to each of the iodine tubes 
5 cc. of water. The characteristic color of the starch and the 
several dextrines will be more apparent. Complete conversion 
should take place in about 15 minutes. If it does not repeat the 
experiment us4>n& a larger amount of saliva. 
b. To IcO" -dr;rrf S joJu saliva-, S A L I V A. 
32a 
mix and make tests as rapidly as possible wich iodine and. with Feh- 
ling solution. What is the result? 
c) Boil 5 cct of saliva in a tube for 1-2 minutes, then 
add 10 cc. of the starch solution and mix. Immediately test a por- 
tion as above and also at the end of 15 minutes. What is the result? 
What is the action of heat on ptyalin? 
d) . To 10 cc. of starch solution add 0.2 cc. of a if acetic 
acid solution, mix and then add 2 drops of saliva. Tost immediately 
with iodine and with Fehling solution, and also at the end of 5, 10, 
and 15 minutes. The mixture contains about 0.02/i acetic acid. What 
is the effect of this amount of acetic acid on the rate of inver- 
sion? 
e) To 10 cc. of the starch solution add 0.6 cc. of a dilute 
KCl(0.3h). The latter is prepared by adding 10 cc. of the concen- 
trated acid to one litre of water. Mix and then add 2 drops of 
saliva. And again mix thoroughly. This mixture now contains about 
0.02/? MCI, about the same degree of acidity as in the preceding 
experiment, and about .1 of that of the gastric juice. Tost the 
mixture at once with iodine and with Fehlingsolution, and also at 
the end of 5, 10, and 15 minutes. Note carefully the result. Hoy; 
doasthe action of HC1 compare with that of acetic acid. 33 
CHAPTER V, 
GASTRIC JUICE. 
I„ RECOGNITION OP PRES HYDROCHLORIC ACID.- 
Three solutions of dilute HC1 labelled 1, 2 and 3 will be found 
on the side-table. Solution 1 approximates in strength that found 
in the gastric juice. It is prepared by diluting 6cc of HC1 
(1*19 specific gravity) to one liter (=0.25/Z). Solution 2 is 
prepared by diluting 200cc of solution 1 to one liter (=0.05/0. 
Solution 3 is prepared by .diluting 200cc of solution 2 to one liter 
(=0o0i^0 
A 2fa pepton and a !$ lactic acid solution will also be found 
on the side table. If the pepton solution is slightly alkaline 
it should be faintly acidified with acetic ar lactic acid. 
The following tests are given in the order of their delicacy. 
1). Di-menthylamidoazobenzol.—This reagent is used in a 
alcoholic aolution. Add 3-4 drops of the reagent to some of the 
solution to be examined. If a pink red color forms a free mineral 
acid is present. In the case of gastric juice it is HC1. A 
yellow color indicates an absence of HC1. Certain substances 
juch as pepton and organic acids tend to interfer in this as well a 
as in the subsequent tests. 
Note the results obtained with solutions 1, 2 and 3 in , 
first column. Then mix the same amount of these solutions witn- 
an equal volume of 2% pepton and to this mixture apply the test 
and note the results in the second column. In the same way make 
a mixture of the three solutions with an equal volume of a 
solution of lactic acid, test and note the results. 
aa 2% pepton. aa 1$ lactic acirL. 
Icc of Solution 1. : 
lcc " " 2. : 
Icc " *» 3. : 
Limit of delicacy. : 
• « I 
• • ■ 
• • 
m • 
. • 
Apply the test to thersolution of pepton, also tbethe solu- 
tion of lactic acid. Report the results. 
Ql) a Giinzburg&s reaction of the test.— ' 
The reagent is prepared by dJjs^O-lvine lg of vani 1.11 rr-aal-^g.of 
phloroglucin in lOOcc. of aj^cohol. 34 
Plsies the solution to be tested in an evaporating dish add 2-3 
drops of the reagent and carefully evaporate over a small flame 
to drynesso A purple or pinkish-red color indicates free HC}.« 
This has long been considered the most delicate test for free HC10 
Apply this test to solutions 1, 2 and 3 and to mixtures as 
indicated in the table given in experiment 1, Carefully note the 
limit of delicacy of the reaction under he several conditions0 
!abu1ate results as above. 
3 ). BoasJ reagento—This is prepared by dissolving ICg of 
resorcin, 3g of canesugar and 3cc of alcohol in lOCcc of water. 
Place in an evaporating dish the solution to be tested, add 2-3 
drops of the reagent and evaporate over a small flame to dryness. 
If a free mineral acid is present a rose or pink-red color devel- 
op s and gradually fades on cooling. 
Apply this test to solutions 1, 2 and 3 and to mixtures as 
indicated in the table given in experiment 1. Note the limit of 
delicacy and tabulate the results, 
4) Trcpaeolin 00*-- A solution of this reagent is prepared 
by dissolving 0,25 g of the reagent in lOOOcc of water* Instead 
of the solution trcpaeolin papers may be employed. They are , 
however9 not so reliable as the solution since with distilled water 
they sometimes give a. pink color. To some of the acid solution 
add a drop of the reagent (or immerse a strip of the tropaeolin 
paper)o A pink color is due to free mineral acid. If the solutin 
is evaporated carefully to dryness a bluish residue remains, 
Apply this test to the solutions as given in experiment 1 
and tabulate t he re suits * 
5) Congo red papers.—-The color of these papers is changed 
on contact with mineral acids to a deep blue, whereas organic acids 
yield a violet. Immerse a strip of the paper in lec of the solutions 
1; 2 and 3. also lactic acid and distilled water and report the 
results and the delicacy, 
6) c Benzopurpurin 6B papers.— These papers are turned to an* 
intense dark brown color by mineral acids. With strips of this 
paper make similar tests as those given in experiment 5 and report 
the results. 
7) Methyl violet*— A solution of this reagent is prepared 
by dissolving C*5g in lOOOcc of water* the solution to be tested 
add 1-2 drops of the reagent. Free hCl gives a copper-blue color* 
Organic acids yield a violet blue. Apply this test, first to 
some distilled water, and note the color. Finally apply the test 
to the solutions 1, 2 and 3 and compare the results. Also test 
pent on and lactic acid mixtures and-tabulate the results as -under 
experiment l. .GASTRIC JUICE 
II. rPTECTION OP LACTIC ACID. 
Uffelmann’s test.—The reagent is prepared by adding a drop 
of dilute ferric chloride to lOcc of a.2.hfo carbolic acid solution. 
The liquid is colored blue. This color is completely discharged 
by mineral acids leaving a colorless solution, whereas organic 
acids discharge the color and leave a straw yellow solution. 
A 1% lactic acid solution os used. 
1) In each of 3 test-tubes place 5cc of the reagent then add 
to each about 1/2 cc the lactic acid solution. blue is 
replaced by a fetraw yellow color. 
To each of thesb tubes now add respectively an equal volume of 
the HC1 solutions 1, 2 and 3. Note the interference, if any, 
in the lactic acid reaction by the presence of free HG1. 
2) In each of the 3 tubes place 5cc of the reagent, then add 
respectively an equal volume of the HC1 solutions 1, 2 and 3. 
Compare the results with that obtained above with lactic acid. 
3) In oach of 6 tubes place 5cc of an almost coloress solu- 
tion of PeCl3. To tube 1 add lcc of the HC1 solution 1. To 
tube 2 add lcc of the lactic acid solution. To tube 3 add lcc 
of the 2% pepton solution. To tube 4 add lcc of alcbhol. To 
tube 5 add lcc of a 4% solution df canesugar. Tube 6 
remains blank and serves for a companion. Carefully note the 
results. 
It is evident from the above experiments that this test for 
lactic acid is not characteristic. In the first place free HC1 
if present in sufficient amount may interfere; and secondly, a 
similar test is given by a number of substances which may at times 
be present in the stofcach contents. In order to obtain a positive 
test for lactic acid it is necessary to isolate the lactic acid 
from the liquid by extraction with ether. ?he liquid must be 
extracted several times with ether. ether is then distilled 
off, the residue dissolved in water and tested as above. 
III. PEPTIC DIGESTION. 
The following solutions will be found on the' side-table. 
1) A 0.25/ solution of HC1. This solution is the same as 
Solution 1. used in Connection with the tests for freeHCl. 
It corresponds to the normal acidity of the gastric juice. 
2) o A solution of pepsin in water. This-is- prepared by dis- 
solving lg of pepsin in lOOOce- of ustat-erv' GASTRIC JUICE 
3)* A pepsin-hydrochloric acid solution. This is prepared 
by dissolving Ig of uepsir* in 1 liter of solution 1. 
Label 6 tubes and equip as follows: 
1) c Place in tubes 1 and 2 20cc of solution 3 and- about 2g 
of fresh washed fibrin. Too much fibrin should be avoided. 
Place in tube 3 lOcc of solution 3. Immerse in.boiling 
water for about 2-3 minutes; then cool to 40°, and add 2g of .fibrin. 
Place in tube 4, lOcc of solution 1 and about 2g of fibrin.- 
Place in tube 5, lOcc of solution 2 and about 2g of fibrin. 
Place in tube 6, 2.5cc of solution 3 and 7.5cc of solution 1. 
Then add about 2g of fibrin. 
t f 
The tubes thus prepared are placed in an ,inCubator at 40° 
or immersed in a water-bath having that temperature. At the end 
df 15 minutes the tubes are taken out and examined. Observe,that in 
all the tubes, except tube 5, the fibrin has swelled up so that the 
contents of the tube ace solid. Return the tubes to the incubator 
and'examine at the end of every hour for the next three knurs. 
Observe the change that takes place in tubes 1 and 2 and compare • ' • 
carefully with tubes 3, 4 and 5 and with tube 6. ’ * • \ • 
i 
The tubes remain in the incubator till next day. If, however,,. ‘ 
tube 1 is aompletely digested in 2-3, hours it should be treated ; , 
at once according to experiment .>2. Then carefully examine and 
note the condition of each tube. In tubes 1 and 2 and pdssibly- . 
in tube 6 the fibrin hasd&sappeared. A finely granular, whitish • 
or brownish sediment is left. Y/hat is it ? Tubes 3 and 4 are 
about alike. The fibrin is gradually being dissolved by the di- 
lute acid. The pepsin added to tube 3 evidently has been des- . • • 
troyed by boiling. No change in tube 5. 
Return tubes 2, 3 and 4 to the incubator and keep there for . , 
3-4 days longer. 
* 
2) Filter the contento of tube 1. 
/ 
a). To lOcc of the filtrate, in a small beaker, or in a wide 
test-tube or pot, add 8g of powdered Immerse for. 
l/2-l hour in a water-bath at a temperature of 30-35°. Stir with - 
a rod frequently to bring the salt into solution. -When the 'saJLt . ; 
ceases to dissolve,iewhen the liquid is saturated the albumose * 
present will be thrown out of solution as coarse floccules which 
rise to the surface forming a sticky or &limy layer. Transfer* 
the liquid to a filter previously moistened with a little :satur- 
ated (NH J2SO4 solution. . ■‘ ’ . 
Wash the residue with lOcc of saturated sdlutiorV* • .* . 
a1). The clear filtrate contains pepton. Test this., 
solution as follows: i . GASTRIC JUICE 
37 
l)o To a liter of the liquid add an equal volume of strong 
ITaOH, then 1-2 drops of very dilute solution. A pink color 
results. The biuret test is given in the cold by the hydrated 
prate ie; s „ 
?}o To another portion of the filtrate add 1-2 drops of a 
fresh tannic acid solution. Avoid an excess of reagefct. A heavy 
white precipitates forms. 
3). To a portion add one to two drops of dilute acetic acid, 
then a drop or two of potassium ferrocyanide. What does the 
absence of a precipitate mean? 
b?J The (NH/)2SO4 precipitate left on the filter is albumose. 
Transfer to a tube, add distilled water, warm gently and stir with 
a rod till dissolved. Test this solution as follows: 
.1) o Boil the solution. Absence of coagulation shows absence 
of albumen and globulin. 
2} . To a portion apply the I1HG3 and heat tost for albumen. 
3}c To another portion apply the acetic acid and potassium 
ferrocyanide test for albumene. 
'0‘)r With the remainder of the filtrate from tube 1 make the 
f 0110wing tests: 
1) . Heat a portion to boiling. Wha dees the absence of co~ 
agulation mean? 
2) , To a little of the liquid (lcc) apply the biuret cest 
as given above under a*-I. 
Fxactly neutralize the remainder of the solution with dilute 
NaOH and test for albumoses as follows: 
3) To a portion add 1-2 drops of dilute acetic acid and a 
drop or two Sf ferrocyanide solution. If no precipitate forms 
add some NaCl solution according to directions given ' test. 
4) To another portion apply ghe NHO3 and heat test for albumose, 
C>) . to the yellowish liquid obtained in tube 2 add an excess 
of NH4OH0 An orange yellow color results—the Xanthoproteic 
react ion. 
6)0 To another portion of the liquid add 1-2 drops of fresh 
tannic acid solution. Heavy white precipitate. 
3)o After tube 2 has been kept for 3-5 days at 40°, filter 
the contents„ Saturate lOcc of the liquid with (NH4J2SCX4 according 
to the directions given amove. The liquid is cloudy but very 
little albumose is precipitated. Why? GASTRIC JUICE 
filter the saturated liquid and to a portion of the filtrate 
apply the biuret test as given above under a'-l. 
4). The fibrin in tubes 3 and 4, in a few days at 40°, is 
completely dissolved by the acid present. When this occure unite 
the contents of the two tubes and filter. Exactly neutralize the 
filtrate according to directions given in test 22, a/heavy white 
precipitate shows the presence of acid albumen or as it is some- 
times called syntonm. Pepton may also form but will remain in 
solution. 
IV. EXAMINATION OP STOMACH CONTENTS. 
1)* The stomach and contents of a recently fed rabbit (or 
larger animal) are cut up, diluted with about 500cc of water 
and placed at 40° for about 1 hour. The mixture is then filtered 
through muslin. This dilute gastric juice is used for the fol- 
lowing experiments: 
1) * Test the reaction with litmus paper. It is distinctively 
acid. 
2) Test portions of the liquid for free HC1 according to I, 1&2. 
3) Test a portion for lactic acid according to II, 1. 
4) To lOcc of the solution add a shred of fibrin or a flake 
of coagulatedegg albumin. Set aside at 40° for 2-3 hours. 
If not dissolved let the tube remain at this temperature over night. 
Then filter and to the filtrate apply the biuret test in the cold. 
Ill, 2a. 
5) Apply the biuret test direct to a portion of the dilute 
gastric juice and compare the intensity of the reaction with that 
obtained in 4. 
6) In each of 3 test-tubes place lOcc of fresh milk. 
To tube 1 add 2cc of the solution, preciously carefully neutralized. 
To tube 2 add one drop of commercial rennet solution. Tube serves 
as a control. Bet the tubes aside at 40° for 1 hour then examine. 
2). Test for pepsin.—The following test is applicable to 
vomited matter, or the liquid obtained from a stomach. Dilute 20ccc 
of the liquid, if neccessary, and filter, To one half of the 
filtrate in a test-tube add a few shreds of washed fibrin, or a 
flake of coagulated agg albumin. Set aside at 40° for 1/2-1 hour-. 
The fibrin should dissolve,the egg albumin requires more time. 
If no digestion takes place it may be due to the absence of KOI, 
or of pepsin or of both. 
To the other half of the filtrate add an equal volume Q+5% 
HC1* This is prepared by diluting 6cc pf concentrated HC1 (1,19 
specific gravity) with water to 500cc. GASTRIC JUICE 
To the mixture of filtrate and ads I lire* in or egg albumin 
and set aside at 40° as above. If in both these tests the fibrin 
or albumin remains undissolved it is due to the absence Of pepsin* 
Each student will receive five "uhknowns" and these are to be 
tested for lactic acid, free HOI, and for pepsin. Report the 
results• 
Pepsin is not present in the gastric gluid in atrophy of the 
mucous membrane of the stomach. 
t / . 
"The flakes of coagulated egg albumin are best prepared by 
gradually pouring a dilute solution of the egg albumin, with constant 
stirring, into boiling water. 39 
CHAPTER VI * 
PAN CREST IC SECRETION. 
<3ut up the fresh pancreatic gland into very fine pieces, 
or better pass it through an Enterprise fruit-press. The pulp 
thus obtained can be used direct, or mixed with several volyiqes of 
water. 
Place about lOcc of the pulpy mixture in a small beaker, 
add 25cc of water and boil for about 10 minutes. ©rush the hard, 
coagulated lumps in a mortar and return to the liquid. Reserve 
this for experiment 1. 
1). Cleavage action on fats.—The fat or oil employed for this 
test should be strictly neutral. It can be obtained in this con*- 
dition by the following process: Place about lOcc of the oil 
(cotton-seed oil or butter) in a small separatory funnel, add 20cq 
of water and render the mixture distinctly alkaline with NaOH* 
Then add an equal volume of ehher and shake till the fat dissolves. 
Draw off the aqueous liquid and to the ether add an equal volume 
of waterand shake again to wash the ether. Remove the aqueous 
layer and wash once more with water. Transfer the ether solution, 
filtered, if need be, to a porcelain dish and allow the ether to 
evaporate. The neutral fat is left behind. 
Place in each of two test-tubes about 5-4 cc of the heutral 
fat, 15cc of water and a few drops of concentrated aqueous blue 
litmus solution. 
a) . To one test-tube add about 5oc of the fresh pancreatic 
pulp mixture and shake. 
b) To the second tube add one half of the liquid containing 
the boiled pulp and shake. If the contents of the two tubes 
react acid add, drop by drop, a NagCOs solvit ion until the 
mixture is distinctly alkaline. 
Place the tubes in an incubator at 40* for 6n8 hours, or over 
night* Compare the reaction of the tubes, Reserve the two mix** 
tures for the naxt-experiment, If the mixtures repair tOQ Ineg 
at this temperature bacteria develop and,giving rice to ftAJUtS, 
reduce the litmus. The two tubes will then be Uhd 
will give the same me suite in tba jaaxVaxp^rtaetnT. 
Under the inXlu-erio© of a in the pancrt&§i kfiOwn StS 
steapsin, or ptalyn, the neutral fat 
free fatty acid and glycerin. PANCREATIC . 
40 
2) Emulsifying action on fats.—After digesting the two mix- 
tures at 40° for 6-8 hours in the preceding experiment, shake thonroug 
ly and take * l/s—1/2 of the contents of each tube andtreat 
as follows: 
a) To a portion of the mixture from Exp. la. add about lcc 
of solution (2%) and shake thoroughly. The liquid becomes 
milky ana on standing the fat does not rise to the surface. 
Examine a drop of the emulsion under the microscope. 
b) . To a portion of the mixture from Exp. lb. add Na2C0s 
solution as above and shake. The liquid does not emulsify. 
The fat rises rapidly to the surface*on standing. If bacterial 
decomposition has taken place, or if the fat was not neutral in the 
beginning, some emulsification will result. 
Why is the fat emulsified in one case and mot in the other? 
3) Diastatic action on carbohydrates.— Prepare a starch 
paste by boiling lg of powdered starch with lOOcc of water. 
Place in each of two test-tubes 15cc of the starch paste. 
a) o To one add about 2cc of the pancreatic pulp mixture 
mix andplace in a water-bath at 40°. 
b) To the second add 1/4 of the boiled pulp . mixture and like* 
wise set aside in the water-bath at 40°. 
At intervals of about 15 minutes take out a portion, about 
2cc, from each of the two tubes. Test one half of each portioh 
with iodine for starch and dextrine. Test the remainder for sugar 
by boiling with Pehling’s solution. How soon does dextrine make 
its appearance? How early can sugar be recognized? When is the 
achromic point reached? 
Note the change in the appearance of the two tubes and the 
difference in results. Compare this action of "pancreas with that 
of sAlive. 
4) Proteolytic action.—In a 50cc Erlenmeyer flask, provided 
with a cork, place about 5g of fresh fibrin and about 20cc of 
chloroform water. This is prepared by adding 2cc of chloroform 
to 50cc of water and shaking thoroughly. To the fibrin and chlori 
form water add about 5cc of the pancreatic pulp and mix well. 
Render the mixture distinctively alkaline by the addition of a 
few drops solution Cork the flask and set aside 
at 40° for 2-3 days. 
Occasionally examine the contents of the flask and compare th 
rate of digestion with that of gastric juice. Note that the fibr 
does net swell up as in gastric digestion, and that the edges are \ 
evenly eaten away. 
The contents of the flask .. .are niilly sJJ>glitJ^--a^idailaled P A N C R E A 
41 
with acetic acid, boiled and filtered. The filtrate may contain 
albumose, pepton, tyrosin and leucin. 
a) To a portion of the filtrate ap< ly the biuret test in the 
cold. 
b) To another portion add a few drops of bromine water and 
shake. A pink to a purple-red color (proteinorchrom) develpps. 
This is known as the bromine reaction and is due to an unknown 
substance, proteinochromogen or tryptophan. 
c) Concentrate the remainder of the filtrate on a wathh- 
glass to a small volume (a few cc) and set aside in a cotbl place 
over night. Examine the deposit with the microscope for tyrosin 
which forms characteristic bundels of needles, and fo,r leucyin 
balls* If no deposit forms concentrate to a thin * 1 syrup 
and again set aside for examination. 
L E U C I N . C6H13N02. 
This compound was formerly considered to be x-amidocaproic 
acid but the more recent studies of Schulze and Likieruik have shown 
it to be x-amido-isobutyl-acetic acid-- (CH^)gCH.CH2-GH(NH2).COgH. 
Leucine is readily formed and is a constant cleavage^procuct, 
in the decom;osition of proteids, gelatin and horn. This decom- 
position occurs in pancreatic digestion; may be brought about by the 
actions of acids and alkalies at high temperatures; and may also 
occur as a temporary bacterial product during putrefaction. 
Plant proteids, as well as animal proteids, can give rise to leucin 
It can be readily prepared from proteids, or better white horn, 
by boiling with dilute HC1. Leucin has been found, in diseased 
conditions, in'various organs and glands of the body, in pus, 
blood, and in decomposed epidermis such as is found on the feet 
and between the toes. In the latter case the peculiar Oder is larg 
ly due to decomposition products of leucin. This compound occurs 
with tyrosin, in the urine in liver diseases, especially in acute 
yellow atrophy. 
Leucin j.s ' dextro-rotatory in acid or in alkaline 
solutions but in neutral solutions is inactive. On heating with 
baryta it becomes inactive. By the action of penicilldum 
the latter variety is changed into a laevo-rotatory variety. 
Several isomeric leucins have been prepared synthetically. 
In the pure condition leucin forms glistening white plates, 
which do not readily moisten when touched with water. As usually 
met with, however, it forms balls or aggregations of spherical 
bodies which often showslight radial marking and are faintly 
refractive to light. When impure leucin is more readily solu ;ble 
than when pure. It is readily soluble in cold water (27 parts); 
mere readily in hot water. It is difficultly soluble in aloohol, 
but is readily soluble in acids and alkalies. It forma aaIts 
with acids and bases. Liunn, 
42 
’The following experiments are made with leucin furnished by the 
laboratory: 
1)« To a drop of water on a slide add a little leucin, about 
the size of apin head. Observe the behavior on contact with wa£e-r« 
Then mix, place on cover-glass and examine under the micro&oope# 
Sketch the crystals observed. 
Then add a drop of water to the edge of the cover-glass and 
gently heat over a flame until the leucin dissolves. Set aside to 
ccol slowly, then examine and sketch the crystals. 
2*) To a few cc of urine in a watch-glass add a little leucin, 
mix and head till it dissolves. Concentrate on the water-bath to a 
small volume; cover with a beaker and set aside over night. Then 
place the watch-glass under the microscope and examine with a low 
power. Typical light yellow spherules or mulberry-like masses 
of leucin will be found. Transfer some of the deposit to a slide 
and examine with a higher objective. 
3) To about 1 c.c. of Ttfater in a test tubo add leucin and 
shake till it ceases to dissolve. Divide into two portions: 
a. To one add a drop of HC1, diluted. 
b. To the other add a drop of dilute NH^OH. 
4) Plaae some leucin in a glass tube, about 6 inches long, 
and open at both ends, and heat gently in an inclined position. A 
portion of the leucin is sublimed as a woolly deposit. At the same 
time an odor of amylamin is given off. 
5) Dissolve some leucin in a little water and render the so- 
lution alkaline with Na OH. Then add 3l-2 drops of dilute so- 
lution. The cupric hydrate precipitate which forms at first, redie 
solves since v/ith leucin it forms a soluble compound. The solution 
is colored blue and on heating^does not reduce. This action of 
leucin is- similar to that of glycocoll, of tarbrates, and of bile. 
6.) Place in a dry test tube a piece of solid KoH about l/2 
inch long; add some leucin and a drop of water. Heat cautiously 
till the KOH melts. Place a strip of moist red litmus paper near 
the mouth of the tube. What is the result and to what is it duo 
Pour the melted KOH into a small beaker, rinse the tube with a littfc 
water, and add this to the beaker. Place the beaker in cold water 
and add very cautiously, drop by drop, H till the solution is 
acid. Then heat the beaker over a flam# and note the peculiar odor 
of valerianic acid. If the odor is not marked pour the liqui nco 
a test tube, cork and set aside for a day or two. On opening the 
tube the odor will be perceptible. 
7) Place some crystals of leucin on a platinum foil, add a 
drop or two of nitric acid (1.20 specific gravity) and evaporate car.- 
fully to dryness. A colorless scarcely visible residue remains. N* 
add a few drops of MaOH and warm. The residue dissolves forming c 
ilear or slightly colored solution. On cautious concentrate, n 
illy drop remains which does not moisten the foil but tlo, 
This is Jcnown - .as Scherer's test and is very 
For the detection of leucin in urine see Tyrosin. TYROSIN. 
43 
TYROSIN, CqH NO,. 
9 11 5 
Tyrosin has been prepared synthetically and is therefore known 
to be para—oxypheny1-alpha-amidoprApionic acid, 
OH 
o 
ch2.ch(nh2).co2h. 
Tyrosin is a constant cleavage product resulting from the ac- 
tion of tyypsin, bacteria, acids or alkalis on animal or vegetable 
proteids or horn. It is not obtained from gelatin or gelatin- 
yielding tissues. It is as a rule accompanied by leucin. It is 
present in old cheese and its name refers to this source. It is 
present in the intestines during proteid digestion but is not present 
in the tissues, bood or urine of the normal body. It is met with 
in the urine in phosphorous possoning and in acute yellow atrophy 
of the liver. 
It forms deli cate colorless silky needles which melt at 235°. 
The crystals often group in bundles and when very impure may form 
leucin-like balls. It is very difficultly soluble in cold water 
(1-2400), more soluble in hot water, insoluble in alcohol or ether. 
It is readily soluble in dilute alkalis and in dilute mineral acids. 
In acid solution tyrosin is laevo-rotatory whereas the synthetic re 
duct or that prepared by the aid of alkalis is dextro-rotatory. 
The tyrosin necessary for the following experiments is furnished 
by the laboratory: 
1) • Treat a small portion in the same way as in experiment 1, 
under Leucin. How can . the bundles of fine needles of tyrosin 
be distinguished from similar bundhes of needles of fatty acids? of 
calcium sulphate? 
2) Test the solubility of tyrosin according to the directions 
given in eperiment 3 under Leucin. 
3) To some water in a test-tube add a little tyrosin, then a 
few drops of Millon’s reagent. Heat the liquid till it begins to 
boil. It colors rose-red, and on standing becomes dark-red and 
may yield a red precipitate. This is known as Hofmann*s reaction 
and is due to the presence of the oxy-jphenyl group in tyrosin. What 
other substances give this reaction with Mlllon* s reagent? 
4) Place some tyrosin in a dry test-tube, add a few drops of 
concentrated Place the test-tube in a water-bath and heat 
at 100° for about naif an hour. Then cool and pour the contents 
into a small beaker containing some water. To this liquid now 
add BaCCuin small portions while stirring, until the reaction ceases 
to be acid. Filter the liquid and concentrate the filtrate to a 
very small volume. To this add a drop or two 
of very dilute A beautiful violet color This 
test is known as Piria*s reaction. T Y R 0 S X N. 
5) Place some crystals Of tyrosin on a platinum foil, add 
nitric acid (1.2 sp. g.) and warm. The tyrosin becomes bright 
orange yellow and dissolves# Evaporate very cautiously to dryness 
when a deep yellow, transparent redidue remains. Add a few drops 
of NaoH and a deep reddish-yellow solution results. This on eva- 
poration leaves r>n intense brown residue (Scherer’s test)., 
n 
A similar reaction is given by the other substances and conse- 
quently it is not characteriscic. 
6) To a boiling aqueous solution of tyrosin add some 1 per 
cent, acetic acid and then sodium nitrite solution., drop by drop, 
A beautiful red color develops (Wurster). 
7) a To a hot aqueous solution of tyrosin add some dry quinone. 
The liquid becomes colored a ruby-red (Wurster). 
DETECTION OP LEUdIN AND TYROSIN IN URINE. 
Tyrosin may occur in the sediment in urine but may be in 
solution. Inasmuch as leucin is more soluble it will be, as a rul$ 
in solution in the urine. 
Precipitate the urine with basic acetate of lead, filter 
and remove the lead from the filtrate by hydrogen sulphide. Then 
concentrate the solution as low as possible and set aside to crys- 
tallize. Examine under the microscope for crystals of leucin and 
tyrosin. If leucin is present it can be removed by means of warm 
alcohol• 45 
CHAPTER VII. 
THE BILE. 
flile is a mixture cf the secretion of liver cells and of mucin 
derived from the cells lining the bile-bladder and duct. It is a 
thick, tenaceous fluid and is alkaline in reaction* The specific 
graulty ranges from 1*01 to 1.04. The color of bile varies in differ- 
ent animals. It may be light yellow, brownish yellow, brownish 
green, green, and greenish blue. Human bile is yellowish, at times 
greenish. Bile posssses a pronounced bitter taste. It does not 
coagulate on heating. Human bile contains ture mucin, whereas, ox- 
bile contains but traces of mucin and instead n muceoahbumin. 
The quantity of bile secreted in 24 hours is subject to con- 
siderable variation even in health. In the case of fistulas, from 
to 1 liter of bile has been observed to be secreted in 24 hours, 
but the secretion under these conditions can hardly be considered 
as normal bile. The actual quantity fiven off in a day is probably 
not less than a half a liter. After a proteid diet the secretion 
is increased, whereas, with fath and carbohydrates it is less marked 
The secretion is also decreased in starvation. The secretion is 
continuous but with variable intensity. Inasmuch as the bile 
flows from the bladder under very little pressure a slight obstruct 
tion in the duct may lead to retention of the bile. As a result 
the bile constituents are absorbed and may appear in the urine. 
Human bile as it is found in the bladder after death has been found 
to contain from 7-18 per cent, of solids. The bile as it flows 
from the liver, in a fistula, contains much less solids, 1-4 per 
cent. The bile, therefore, becomes concentrated in the bladder 
by absorption of water„ 
Bile contains as characteristic constituents certain salts of 
bile acids, bile pigments, and small quantities of lecithin, choles** 
terin, soap, neutral fat, urea, and salts of calcium, magnesium, 
iron, and copper. 
Tje bile acids are usually present as sodita salts* In some 
sea-fish they are in combination with potassium. It is customary 
to speak of two bile acids, glycocholic and taurocholic. The former 
on cleavage yields .. glycocoll and cholic acid; the latter 
taurin and cholic acid, Inasmuch as this cholic acid is but one 
of several cholic acids known, it follows that there is a group of . 
glycocholic acids, and a group of taurocholic acids. Human bile 
yields three cholic acids. The bile of some animals may contain 
only glycocholic acid, or only taurocholic acid; whereas, in some 
variable mixtures of the two acids are present. Thus, the feauro— 
cholic acid predominates in the bile of carnivorous animals, birds, 
reptiles, and fish. The bile from the rabbit and the hog contains 
almost entirely Glycocholic acid. Herbivorous animals as a rule 
contain variable quantities of both acids. Both glycocoll and 
taurin are amido acids, Taurin contains S as a characteristic 
constituent. According to Hammersten the bile of some animals 
contain a third group of bile acids which are rich in S and which'in 
their behavior to mineral acids resemble ethereal sulphates. 
The bile—acid salts are precipitated from their solution in 
water cr alcohol, on the addition of other, as Xine- needles. The 
bile acids &fttl their salts- -are dextriw>-natartoryl B-I L E. 
46 
A large number of bile pigments are known but in normal bile, 
as a rule, there are but two, bilirubin and biliverdin. The former 
can be obtained as a reddish yellow powder; the latter as a greenish 
powder. The color of the bile is due to the preponderance of 
| one or the other of these two pigments. Ox-bile has both pigments. 
The other bile pigments as, bilifuscin, biliprasin and bilicyanin, 
! have been isolated from bile stones and altered bile. 
The bile pigments are soluble in alkalis, insoluble in acids, 
! and yield insoluble compounds wjth calcium and other metals. Bili- 
! pubin is slightly soluble in alcohol and in.ether, readily soluble 
I In chloroformo Biliverdin is insoluble in chloroform. Bilirubin, 
jin addition to being in the bile, is met with in bile stones as 
a calcium compound; in old blood extravasations (haematoidin) and 
I In urine and tissues during icterus. 
The source of bilirubin is undoubtedly haematin. On reduc- 
tion it yields hydrobilirubin which is closely related, if not iden- 
tical, trith stercobilin (found in the intestines) and with urobilin 
of urine. On oxidation it yields biliverdin. The amount of pig- 
ment in the bile is usually only a few hundredths of a per cent., 
rarely 0.1 per cent* 
As to the origin of these bile constituents it may be said 
I that the bile acids are elaborated by the cells of the liver, not 
, elsewhere in the body. The bile pigments, without doubt, aan 
be formed in other parts of the body, than in the liver, but undsr 
normal conditions the liver is the organ where they are formed. 
I Taurin and glycocoll result from the decomposition of proteids in 
| any part of the body. 
1) Place some dilute bile (1-5) in a test-tube and heat to 
boiling. Immerse a strip of red litmus paper, then remove and 
I wash with water. The reaction is distinctly alkaline. 
2) Place about 5 ee of bile in a test-tube, add 10 cc. of watr; 
mix and filter if necessary. To the clear liquid add acetic acid. 
A cloudiness or distinct precipitate of mucin or nucleoalbumia 
forms on standing. This is not marked in ox-bile. 
3) Filter the cloudy liquid obtained in Exp. 2, and apply 
the biuret test to the clear filtrate. Absence of proteids. 
Notice also, that the which forms redissolves in 
the bile solution and yiel&a, a blue liquid which on heating gives 
a black precipitate. What is the cause of this black precipitate? 
What other substances., redissolve Cu(0H)g and yield blue solutions? 
4) To about 20 cc. of bile in an evaporating dish add about 
5 g. of animal charcoal and evaporate on the water-bath, with 
frequent stirring, to complete dryness. Transfer.the residue to & 
150 ccc Erlenmeyer flask, provided with a cork and condensing tube, 
q/id about 30 cc. c* absolute alcohol and boil on the water-bath for 
ftbout half an hour. Oool and filter into a dry flask (or a 50cc. 
test-tube on foot). To the alcoholic filtrate add-anhydrous ether 
till a permanent precipitate forms. Then cork and set aside in 
o tool place over night. The sodium salts of glycocholic and tauro- 
oHoXic acids crystallize out. Filter off the crystalling deposit 
save the filtrate. Squeeze the crystals as dry as passible on 
1 ir BILE. 
47 
the filter in the funnel, then dry between several sheets of filter 
paper. Savo the crystals for subsequent tests. 
5o) The alcohol ether filtrate from the preceeding experiment 
contains, among ether things, cholesterin. Place this filtrate in 
an evaporating dish and allow the ether to evaporate spontaneously, 
then cautiously evaporate to dryness on the water-bath. Rub up 
the residue, thorgughly, with some ether, filter the bhher solution 
into a small beaker or watch-glass, and allow the ether to evapor- 
ate spontaneously. Examine the residue under the microscope for 
the characteristic crystals of cholesterin. Catty crystals, in 
the form of needles, are likely to be present. 
5). DETECTION OP BILE ACIDS. 
On the side table are two sets of diluted bile--bile- 
water, bile-urine—1-10, 1-100, 1-500, 1-1000. Apply the following 
tests first to the "bile-water" dilutions, then to corresponding 
dilutions of bile with urine. Tabulate the results. 
Place about 5 cc. of each of these solutions in test-tubes and 
s-PPlv the following test, noting carefully the delicacy of the 
reaction, 
a. To the liquid to be tested add about two-thirds its 
volume of concentrated sulphuric., acid. The acid is allowed to 
run down the side cf the tube slowly, so as not to mix. The tempera- 
ture shouldnot rise over 60-70°. If necessary, therefore, cool 
partly under the hydrant, then add 2-3 drops of a solution of cane- 
sugar (i-10) and tap the tube gently. A pink to a red or violet 
color develops according to the amount of bile acids present. The 
foam which forms on shaking is likewise colored pink.. This is 
known as Pettenkofer*s test, and depends upon the formation of fur- 
furolo An excess of sugar and too much heat must be carefully avoided 
Observe the difference in the delicacy of the reactions in aqueous 
and urine solutions of bile. 
To some wrater in a tost-tube add sulphuric acid as above, then 
about 5 drops of the sugar solution. Notice the yellow to a 
dark-brown color that forms. Repeat this blank test with urinp, 
acid, and 5 drops of the sugar solution. 
b. Purfurol Test.- Since Pettenkofer1s test depends upon 
the formation of furfurol out of the sugar added the former can be 
added direct. 
To a few cc. of the solution to be tested add one drop of a 
loO/if aqueous furfurol solution, then add slowly as in the preceeding 
test about an equal volume of concentrated sulphuric acid, cool 
somewhat, if necessary, and avoid an excess of furfurol. The 
reaction is often less intense than in 6a. 
Apply this test to some diluted bile. Dissolve a little 
of the crystallized bile acids obtained in experiment 4, in some 
water. Observe the foaming of the liquid when shaken. Divide 
this solution into two portions and test one according to Petten- 
kofer, the other with furfurol. 
c)9 Detection of bile acids in the urine (Hoppe-Seyler*s 
Method)o— The test given above under 6a is usually employed. It BILE, 
48 
should be remembered however that substances may be present in ur£ne 
which will give a reaction similar to that of bile acids0 Moreover, 
in highly colored urines the reaction can be readily masked* In 
such cases the following method of Hoppe-Seyler, though somewhat 
long, will give good results. About 100 cc, of the urine is eva- 
porated to a syrup and the residue extracted with strong alcohol* 
The alcoholic filtrate is evaporated to dryness, and the residue 
obtained is dissolved in water. The qqueous solution is precipi~ 
taged with load acetate and ammonia. The precipitate is wahed, 
then transferred to an evaporating dish or flask and extracted 
with boiling alcohol. The alcoholic solution is filtered while 
hot® A few drops of soda solution are added to the filtrate and 
this is then evaporated to dryness. 
The dry residue can how be dissolved in water, the solution 
slightly acidulated with Hp.8O4a.nd filtered. Tho qqueous filtrate 
can now be tested directly for bile acids according to Ga or b* 
7). DatgSTIOW 0? BILE PIGMENTS. vt 
On the side-table will be found five 
bottles con banning urine di3.uted with bile in the following pro- 
portions :?1-10, 1-20, 1-50, 1-100, 1-500. Apply the following tests 
to these solutions and tabulate the results. 
a). Gitalin* s Test .-Place some bile on the suspected urine 
in a small evaporating dish and add a drop or two of fuming 
a play of colors, green, blue to violet results. With ox-bile the 
colors -are weak and rapidly change. In urine the green color is 
especially important since indican may also give a blue color. Va- 
rious modifications of this test have been suggested and of these 
the following are especially useful. 
a*. Filter the bile solution or suspected urine. Then 
add a drop or two of fuming nitric acid to the moist filter paper0 
The colored rings are very distinct* This test (Rosenbach3s) 
is much more satisfactory than the preceding and is especially use- 
ful when the urine is highly colored. 
2 
a o) To a few cc. of fuming HNO* in a test-tube add slow- 
ly some dilute bile solution or the suspected urine so that the two 
liquids do not mix, Colors develop at the zone of contact. Final- 
J-U mix the contents; a decided green color forms, especially cn 
standing, 
b. Huppert'a Reaction.— To about 10 cc. of the diluted 
bile or suspected urine add a little calcium chloride, then an 
excess of ammonium or sodium carbonate. The billtubin—calcium 
compound is precipitated. Filter, wash the’precipitate then 
transfer while moist to a test-tube and fill it half full of alco- 
hol which has been acidulated with sulphuric acid* Immerse the 
tube for 10-15 minutes in a water-bath, heated, so that the contents 
of the tube are kept near the boiling point. The solution be- 
comes colored an emerald to a bluish green. Now cool the conten- 
ts of the tube, then add fuming HN0_. The green color changes to 
blue, violet, and red . This very delicate and is especial 
ly useful when the-m ine is highly* colored, or .contains-much jindican 
or biood p igment,s , BILE. 
49 
c. Iodine test.— Place the diluted bile or suspected urino 
om a test-fcube, incline the tube and add cautiously 2-3 cc. of 
a dilute tincture of iodine so that it forms a layer. Immediately 
or after a few minutes a bright green ring forms at the zone of 
contact (Rosin-Smith). This reaction is almost as delicate as 
that of Huppert. 
d.) Jolle's Test.- This is claimed to be the most delicate 
test for hile pigments. Place 50 cc. of the suspected urine or a 
mixture of bile and ur ing (1-500) in a glass stoppered cylinder, 
add a few drops of 10/? then BaCl0in excess and 5 cc. of chlo- 
roform and shake vigorously for several minutes. Set aside for 
about ten minutes for the precipitate and chloroform to settle. 
Transfer the chloroform and precipitate by means of a pipette to 
a test-tube. Immerse the tube in a water-bath having a temperature 
of about P°°. The rh\t -oform evaporates in about 10 minutes. 
Remove the test-tube and after a few minutes when the precipitate 
has bottled decant the supernatent liquid. The precipitate is 
colored yellow if bile pigment is present. Allow 3 drops of 
concentrated ( to ?/hich about 1/3 fuming been added) 
to run down the side of the tube. The characteristic play of colors 
develops. 
e.) Acidulate some dilute bile with acetic acid, add a few 
cc. of chloroform and shake. The chloroform dissolves the bilirubin 
and is colored yellow. 
BILE STONES. 
The calculi found, most often in the fall-bladder of man consist 
chiefly of cholesterin. They may he grayish or yellowish white, 
wax-like in appearance, or may be colored from a light red to a 
dark brown. The color depends upon the amount of bilirubin 
present. This pigment is not free but in combination with calcium* 
The number of stonespresent in the gall-bladder may vary from a 
few to several hundred. The size will, therefore, vary considerably, 
from that of a grain of wheat to stones from l/2-l inch in diameter. 
As a result of friction the stones frequently show smooth triangular 
faces. The larger stones when cut in two and polished show generaly 
a concentric arrangement. When they consist of pure cholesterin 
the stones will float on water. Small amounts of fat may also be 
present. 
The bile-stones as usually found in the gall-bladder of cattle 
consist largely or wholly of the calcium-bilirubin compound. Sim- 
ilar c&lculi are met with occasionally in man. These pigment 
stones may contain metals such as iron and copper and even at times 
zinc and manganese. Unlike the cholesterin stones they 
are always heavier than water. 
\ 
A third form of bile-stone very rarely found in man consists 
chiefly of calcium carbonate and phosphate. 
EXAMINATION OP BILE STONES. 
Pulverize a small bile-stone and pla ce the powder in a test- 
tube. Add a mixture of alcohol and ether, equal parts, and warm, 
gently until the powder ceases iro dissolre „ Decant- tinr ethoi BILE 
50 
alcoholic solution onto a watch-glass or evaporating dish and allow 
it to evaporate spontaneously. If the crystals are imperfect 
redisolve in hot alcohol and allow the solution again to evaporate 
spontaneously. 
Save the crystals for the subsequent tests for cholesterin. 
If these is a residue insoluble in the ether-alcoholio mix- 
ture add to it some dilute HC1. An effervesence indicates a 
carbonate (CaCOg). If an insoluble residue still remains wash 
it with water and examine for iaile pigments. 
Evaporate the HC1 solution to dryness and ignite; then dis- 
solve the residue in dilute HC1 and add A blue color in- 
dicates the presence of copper. 
Cholesterin, C27H45QH. — is a common constitutnt though in 
minute quantity of the normal fluids and tissues, of the body. 
Under pathological conditions i.t is met with especially in bile 
stones. It is Also present in atheroma nodules, in tubercular 
masses, tumors, sputum, pus transudates and cystic fluids. 
It is rarely present in the urine and then in small amount. 
A rare urinary cholesterin calculus has been reported by Horbues- 
ewski. Compounds closely resembling cholesterin, possihly isomers, 
are found in plants (phytosterius). 
Cholesterin forms white, glistening crystals which under the 
miscroscope appear as evry thin transparent plates with a corner 
more or less notched. The crystals melt at 145° whereas plant 
cholesterin melt'- at 155°. It is insoluble in water, in dilute 
acids, and in alkalis. It is readily soluble in boiling alcohol 
from which on coiling it recrystalizes. It is readily soluble in 
ether and chloroform. 
1) Examine under the microscope and sketch the characteristic 
crystals of cholesterin obtained from a bile stone. 
2) . To some crystals on a slide under the miscroscope add 
a drop of dilute H SO,, (5 parts of acid to one part of water) 
The edges of the crystals show a bright carmine red color which 
changes to viftlet. 
3). To some crystals as in Exp. 2, add a drop of dilute HgSOa 
then a drop of iodin solution. The crystals turn gradually violet* 
bluish green, then blue. 
4) . Dissolve a few crystals in a little chloroform in a dry 
test-tube/ then-add an equal volume of sulphuric acid and shake. 
The chloroform becomes blood red, then cherry red and purple. 
The acid liquid shows a green flcureseaace (Salkowski). The color 
of the chloroform is quickly discharged if it is poured into a 
moist tewt«tubj, 
5) , Dissolve Bomft cirolestreriA in J2cc of chloroform* a<ULJiQ drops Bill 
51 
Of dfcetic anhydride and then drop by drop* concentrated HgSO.* 
The mixture becomes red, blue and finally green (Liebermann*s 
Cholesterol reactionT. 
6* To a little cholesterin in an evaporating dish add a few 
drops of HC1, and a drop of very dilute On evaporating 
to dryness a blue color results* 
7), Place a little of the dry cholesterin in a dry test-tube 
and add 2-3 drops of propionic anhydride and carefully heat Over 
a small flame till melted. On gradually cooling the mass becomes 
violet then green, blue and red. 
Detection of cholesterin in urine.-- * 
Inasmuch as cholesterin is lighter than water it will be found 
whan present in urine, floating on the Lurface as a thin pellide. 
Some crystals may be dragged mechanically to the bottom. A micrd* 
scopie examination will ofter decide the nature of th&s film. 
This is also true of transudates and other pathological fluids 
where the crystals are often well formed. In the absence of 
typical crystals it will be necessary to employ the following method* 
Extract the urine with ether which takes up fat and cholesterin* 
Remove the ethereal layer and allow it to evaporate spontaneously* 
Examine the residue under the microscope for the characteristic 
crystals of cholesterin. If there is any doubt owing 
presence of fats these must be removed by saponification. 
For this purpose dissolve the residue in hot alcohol, add some 
sirrong alcoholic solution of sodium hydrato and heat on the water- 
bath for some time. Finally evaporate to dryness, and extract 
the residue of soaps with ether. This ethereal solution on evap- 
oration will now give a residue free from fat. CHAPTER VlII, 
BLOOD 
‘ I» MICROSCOPIC EXAMINATION, 
1) * Examine a drop Of fresh blood under the miCrdsoOpe, * 
Measure the diameter and Sketch the red ana white blobd sells, 
What is the difference between the blood cells of mammals and of 
birds, reptiles, etc, 
2) Dilute some fresh blood with water and examine before. 
Observe and sketch the ererated blood cells* 
♦ 
II. SPECTROSCOPIC EXAMINATION* 
1)* Add lec of dafibrihated blood to 50cc of destille4 water 
and shake thoroughly* Place some of the dilute blood in a tevt- 
tube and suspend this about an inch above the slit of the Speotfo* 
scope (Position No. 1.) The test-tube should hot be more than 
one half inch in diameter. A fish-tail burner placed about 3 
inches from the slit serves as a source of light* 
Observe the two absorption bands of Oxy-haemoglobin and thfcir 
post ion on the acale in the spectrum. 
Place in the flame a platinum wire previously dipped in 
a solution of sodium chloride. Notice the characteristic yellow 
line of sodium, its postion on the scale and its relation to the 
two 'absorption bands. 
2 . Now swing into postldon the little outside prism of glass 
so that it shuts off the lower half of the slit. Place a light 
about 3 inches in front of the left face of this prism* The sp<Krtrum 
of haemoglobin appears in the lower half while superposed abovd 
it is a clear spectrum. Place a tube of the blood, diluted and 
well shaken as above, between this second light and the left face 
of the prism (Position No. 2.). The spectrum from this tube Is 
now thrown above that from the tube in front of the slit* 
The two spectra of ohy-haemoglobin coincide. 
a). To tube No. 1 before the slit, add 1-2 drops of freshly 
prepared ammonical ferro-taitrate solution (Stokes1 solution)* 
and examine at once. The two bands of ohy-haemoglobin sooft dis* 
appear giving place to the single wide band of reduced 
Compare this spectra with the superposed one ox ohy-naeWBgipBlni '* 
/fote the change in the color of the tube. BLOOD 
53 
of ferrous sulphate and 3 parts of tartaric acid in water, then 
render alkaline by addition of NH^OH. 
b). To tube $o, ,2.. the one on the left, now add 5-6 drops of 
strong ammonium . sulphide and examine. Ina few minutes 
the single band of reduced haemoglobin takes the place of the two 
bands of , oxy-haemoglobin. The spectra of the two tubes now 
conincide. 
3) To the tube of reduced haemoglobin in position No, 1 
obtained in experiment 2a, add a few drops of concentrated NaOH. 
The single absorption band beconmes replaced by two bands, resem- 
bling those of oxy-haemoglobin but shifted a little to the right. 
The left band is the darker of the two. On standing a few minutes 
the spectra increases in intensity so that the two bands merge 
together; in that case dilute with an equal volume of watdr. and 
examine again. 
This spectrum is due to haemochromogen or reduced haemoglohin 
This test should be resorted !6"when He <nsip60tJ^m~ o 11 KdemoglokTH 
is doubtful. 
Compare this spectrum with the superposed spectrum of reduced 
haemoglobin (2b) . Then substitute for the latter a tube of the 
diluted, well shaken blood, thus placing the spectrum of 
globin above that of haemochromogen. 
4) Dilute some defibrinated blood with about 15 parts of water 
and shake well. Place some of this solution in a test-tube, 
in postion 1. Superpose the spectrum of oxy-haemoglobin using 
dilute blood as in experiment 2 (1-50). The .upper spectrum of the 
very dilute blood shows the two bands of oxy-haemoglobin, whereas, 
the lower spectrum, tube No. 1 which contains a strong solution, 
is entirely dark to the right of the soldium line. 
To the tube in front of the slit, position 1, now add 1-2 
drops of a fresh, concentrated solution ofpotas.&iuai ferrocyanide, 
ffhe color of the liquid changes to a brown and the spectrum of 
Methaemoglobin appears. An intense dark band in the red with 
"two Tess dark bands in the rignt. If the liquid is too concan** 
trated dilute 1/4—l/3 with water. 
♦ 
To the so,ution of Methaemoglobin add a few drops of 
The color and spectrum of oxy-haemoglobin reappears, and in a short 
time this gives way to that of reduced haomoglobinC2bJ . 
5) . about lOcc of concentrated H2SO4 in a test-tube add 
about 5 drops of hlood. Shake thoroughly after the addition of 
each drop of blood ajjid keep the contents of the tube cool. 
Note the dark wine red color of the solution. 
THi7T+_o £> rvorti An nf t.h'tfi. 1 i_aui4 With 2~vi parts*- blood 
54 
cool and examine before the spectroscope (position l) for the 
spectrum of haemotoporphyrin. Superpose as in experiment 2 
the spectrum of oxy-haemoglobin (1-50) for comparison. Hoe mot 
prophysin shows a dark narrow band to the left and a wider, 
darker band to the right of the left band of oxy-haemoglobin. 
Haemotophophysin, is derived from haematin by the 
splitting off of iron. ~Lit results also from the action of 
HBr on haematin. It is an isomer of bilirubin and has been m@t 
with in urine. 
6) To 5cc of diluted blood rl-15) add 2cc of concentrated 
NaOH. The color changes to a cherry rer. Now heat the tube till 
the color changes to a brownish green. Examine before the spec- 
rbscope, position 1, for the spectrum of alkaline haematin. 
If necessary dilute the contents of the tube l/4-~l/3 with water* 
Alkaline haematin shews a dark band through the middle of which 
casses the sodium line Superpose the spectrum of oxy-haematin 
and conpare the two spectra. Then convert the upper spectrum into 
reduced haemaglnhin as in experiment 2a and again compare. 
7) Pass a current of illuminating gas some diluted blood (1-50)„ 
a) Place a tube containing some of the blood thus treated 
before the spectrum in position 1. Superpose the spectrum of 
oxy-haemoglobin (1-50). The lower spectrum, due to carbon mon- 
oxide haemoglobin, is nearly the same as that of oxy-haemoglobin* 
’The two" bands, however, are darker and are removen a trifle to'the " 
right no that the two spectra are not exactly continuous. 
Compare the color of the two tubes. 
b) . Now add to each tube 1-2 drops of Stokes1 solution. 
Carefully note the change in color of the two tubes and also .the 
change in the spectra. 
c) . Again superpose the spectrum of oxy-haemoglobin above that 
of CO-haemoglobin Then add to each tube 5-6 drops of strong 
(NH4)2S2. Examine at once and after the lapse of about 5 minutes- 
d) . Again superpose the spectrum of oxy-haemoglobin above that 
of CO-haemoglobin. Then add to each tube, one drop of a frechly 
prepared strong solution of potassium . ferricyanide. Exam- 
ine at once. The oxy-haemoglomin spectrum changes in a few sec- 
onds to that of methaemoglobin whereas the spectrum of CO-haemoglobin 
persists and is changed only after the lapse of several minutes* 
Owing to the dilution bhe spectrum of methaemoglobin will be faint*- 
CO-haemoglobin is a much more stable compound that oxy-hae- 
moglobin and for that reason the color and the spectrum of the 
solution in experiments b, c, and d, will change slowly, if at all* 
whereas oxy-haemoglobin is readily changed to reduced haemoglobin 
in experiment e b and. and to iD»thaajmoslobija.,in, *d. BLOOD 
55 
DEFIBRINATED BLOOD. 
III. GENERAL REACTIONS. 
1) . Test the reactions of some fresh defibrinated blood. 
a) o Dip a moist red litmus paper for a few seconds into the 
blood, then wash at once in water. 
b) . Place a drop of aqueous red litmus solution on a porous 
porcelain plate. When this has apply a drop-of blood 
to the spot and allow this to remain for about a minute. Then 
wash off with water. Owing to the coloring matter in the blood 
this method of testing is much more delicate that the pre&eding. 
2) To some water in a test-tube add a drop or two of blood 
and mix. Then add tincture of guajac'till the liquid becomes 
cloudy, and finally add some old oil of terpentine. A blue color 
develops at the zone of contact of the liquids and is due to the 
oxidation of t&e guajac. The reaction fails with fresh ail 
of terpentine owing to the absence of ozone. 
a). This test may be applied to urine, suspected of containing 
blood, in the following manner: Place in a test-tube equal volumes 
of guajac and old oil of terpentine. The mixture must not be 
blue. Now add the urine cautiously so that it forms a layer. 
If blood is present a bluish-green ring will form at the zone of 
contact. This is known as Almen’s Guajac Test. The urine if 
alkaline should be neutralized' or rendered faintly acid. Pas 
may give the test with guajac alone. 
3) To 2cc of fresh blood in a test-tube add, without shaking, 
2-5cc of hydrogenperoxide. Oxygen is liberated abundantly and the 
liquid foams and the haemoglobin is gradually decomposed. 
This is due to a so called catalytic action. 
4) To some fresh diluted blood (1-5) is a test-tube add ether 
and gently agitate. The liquid becomes transparent because 
of the solution of blood cells—laky blood. 
5) Pass a current of illuminating gas for a few minutes 
through some dilute blood (1-50). Notice the cherry-red color 
of the solution. As shown above in Exp. II 7, CO-haemoglobin 
is a much more stable compound than oxy-haemoglobin. The following 
guests fetill further serve to demonstrate this fact and are of great 
value in distinguishing between the two forms of haemoglobin. 
The tests c and d are especially adapted for the detection of email 
amounts of CO-haemoglobin in blood. 
a). In one test-tube place aome -dilute ia-anothor BLOOD 
56 
some of the CO-haemoglobin solution (1-50). To each of these 
solution add half a volume of strong NaOH solution (1.34 specific 
gravity). The pure blood solution becomes brownish (due to haematinj 
whereas the CO-haemoglobin solution is unaltered and retains its 
cherry-red or pink-red color (Hoppe-Seyler). 
b) 0 Place 5cc of the diluted blood (1-50) in a test-tube. 
In another tube place 5cc of the CO-haemoglobin solution (1-55). 
To each tube add an equal volume of fresh, saturated KgS-water 
and shake. The pure blood changes to a green, due to the formation 
of sulphur-methaemoglobin, whereas the color of the methaemoglobin 
is unchanged of fades slowly. 
c) . In one test-tube place 5cc of the dilute blood (1-50); 
in another tube 5cc of the CO-haematin solution. To each of the 
tubes add 1-3 drops of dilute acetic acid, then one drop of potas- 
sium ferrocyanide solution (1-5). The proteids in both solutions 
are precipitated but the precipitate in the tube of pure blood is 
brownins, in color, whereas that in the CO-haemoglobin tube is pink. 
On standing a while the pink color changes and both precipitates 
are then alike. 
d) . In one test-tube place 5cc of the dilufceblocd (1-50); 
in ahother 5cc of CO-haemoglobin solution (1-50). To each of these 
tubes add an equal volume of freshly prepared lfi solution of tannic 
acid. The proteids are again precipitated. preci=itate in 
the tube containing pure blood is colored or grayish brown whereas 
that In the Co-haemoglobin tube is pink. An excessof tannic 
acid may dissolve the precipitate and should therefore be avoided. 
Make a mixture of ice of CO-haemoglobin solution (1-50) 
and 4ce of oxy-haemoglobin solution (1-50) add an equal volume of 
the tannic acid solution and compare with the two tubes obtained 
above. 
Haemoglobin is readily decomposed on heating with acids or 
alkalis into globulin and a pigment. If oxy-haemoglobin is acted 
upon the pigment that results is haematin, whereas with reduced 
haemoglobin the product is haemochromogen. The latter decompo- 
sition has been studied in experiment II 3, whereas the formation 
of haematin has been observed in II 6 and III 5a. Haematin com- 
bines with HC1 'o form haemin. 
6)« Preparation of haemin crystals.—place in a small Erlen- 
meyer flask (about 3Qcc. capacity) provided with a cock and conden- 
sing tube, lOcc of glacial acetic acid and heat to boiling on the 
water-bath for about half an hour. Then add, gradually and with 
constant stirring, 3cc of defibrinated blood. Continue heating 
on the water bath for half an hour. / Transfer to a small narrow 
beaker or test-tube and set aside night. Examing 
;he crystalline deposiC microscopically and sketch the form og the 
haemin crystals. BLOOD. 
To preserve the specimen* decant the acetid acid;, then add 
10-20 cc. of water, stir thoroughly and place aside to settle. 
Decant off the water and wash in a similar manner with alcohol; then 
stir up the crystals with ether and transfer tl a small filter. 
Press the crystals between filter paper till dry, then transfer to 
a specimen tube. 
The operation of washing can be greatly simplified by the uS*> 
of a centrifugal apparatus. 
The recognition of fcaemin crystals 16 of the greatest import- 
ance in the identification of blood stains. Each student will 
receive a piece of fabric and a piece of wood stained with blood# 
These are examined in the following manner: 
a) Scrape a little of the stain off the piece of wood. 
Place the scrapings on a glass slide, add a drop of 1% solution of 
NaOl and warm gently over a very small flame, avoiding ebullition* 
until the water is nearly driven off. Then, while still moist add 
1-2 drops of glacial acetic acid, cover with a cover-glass and again 
warm gently over a small flame till most of the acetic acid has 
evaporated. When cool, examine under the microscope for the charac- 
teristic light brown haemin prisms. Sketch the form of the perfect 
crystals. 
b) Soak the cloth in a 1 per cent, solution of NaCl in a 
watch glass and squeeze out the coloring matter as thoroughly as 
possible. Concentrate the liquid, if it is but weakly colored, 
on the water-bath to a small volume. Then place 1-2 drops of the 
liquid on a glass slide, warm gently, as above under a* Until the 
liquid is nearly evaporated then add 1-2 drops of glacial acotic 
acid, cover with glass and again heat gently till most of tho acetic 
acid has evaporated. Cool and examine for haemin crystals. 
7) The formation of haematin and of haemin crystals may be 
utilized for the detection of a small amount of blood or blood pig“ 
ment in the urine. To the suspected urine add NaOH and boil. The 
earthy phosphates are precipitated and are colored brownish red 
by the haematin (Heller’s test). If there is doubt as to the nature 
of the coloring matter in the .precipitate, this can be filtered off 
and subjected to the haemin test according to the directions given 
above under 6a, 
a). The urine may be precipitated with tannic acid and the 
precipitate can then be treated for haemin crystals as above. 
8) Place about 20 cc. of dofibrinated blood in a beaker-and 
add about 200 cc. of water. Acidulate very slightly with acetic 
acid, boil and filter. The filtrate should be water-clear. 
Notice the brown color of the coagulum. To what is it due? 
Evaporate the filtrate to a small volume, about 20 cc. If a pre- 
cipitate forms during the evaporation it should be filtered off. 
Test the clear concentrated liquid as follows: 
a) Roil some Fehling’s solution in a test-tube, then add some 
of the liquid and boll again. A yellowish red precipitate of 
cuprous oxide indicates the presence of sugar, 
b) . Acidulate a little of the liquid with and add some 
AgNOj . A hoavy white precipitate soluble in indicates- the BLOOD. 
57 
presence of NaCl. 
c) .Acidulate another portion with KNO and add some ammonium 
molybdate solutions. On gently warming a^yellowish precipitate or 
coloration indicates phosphates. The test for phosphoric acid 
can be made by adding NH4OH to the liquid, then magnesia mixture* 
A white cloud or precipitate forms ff phosphoric acid is present. 
d) o Evaporate the remainder of the liquid in a watcheglass 
on a water-bath till only a few drops remain. Then set aside to 
cool and examine under the. microscope for crystals of NaCl. 
BLOOD SERUM. 
IV. PREPARATION OP BLOOD SERUM. 
The blood is received, directly from an animal, into a wide 
cylindrical vessel or into a common fruit-jar. It clots in a 
short time forming a solid coagulum. The vessel is then placed in 
an ice-chest for 36-43 hours. As the clot shrinks the clear yelluw 
serum is squeezed out and collects on the top. This yellow serum 
is removed with a pipette and is used for the following experiments. 
It net infrequently happens that the serum as obtained is reddish 
due to the presence of blood corpuscles. In that case it is best 
to place the serum in a tall, narrow beaker and set it aside in the 
ice-chest for 1-2 days when the corpuscles will subside and leave 
a straw-yellow, clear serum above. 
Blood plasma, the liquid portion of the living blood, contains 
at least three protaids~-fibrinogen, serum albumin, and serum globu- 
lin. In the process of clotting the fibrinogen is changed to 
fibrin and hence the blood serum contains the two proteids serum 
albumin and serum globulin, or paraglobulin. 
Carefully review in this connection the work done on the proteids 
of blood serum ( ). What is precipitated if blood serum is 
saturated with With (NH^JgSO^? 
1) Determine the coagulating point of undiluted blood serum 
(5-10 cc.) according to the method given under egg-albumin, Exp.21. 
Noto the temperature at which the contents of the tube become cloudy; 
when they gelatinize and when they become solid. 
2) In each of three tubes place one cc. of blood serum. To 
tube 1 add nothing. To tubes 2 and 3 add 5 and 10 cc. respectively 
of distilled water. Immerse in a boiling water-bath for 10 minutes 
Note the result. No. 1 coagulates solid whereas Nos. 2 and 3 do nfct. 
Sufficient dilution of serum with water renders it non-co-agula- 
ble by heat. If tepid-water is used, owing to the presence of 
calcium salts, partial coagulation will take place. 
3) To each of 4 tubes add 1 cc. of blood serum; then add to 
each 10 cc. of distilled water. To tubes 1 and 2 add 1 and 6 drops 
respectively of a acetic acid; to tube 3 add a couple of drops of 
CaClgSolution; to tube 4 add 2 g. of NaCl. Immerse the tubes in a 
boiling water-bath for 10 minutes. Note the results and explain the 
same. 
As shown above in experiment 4 blood serum diluted with 10 
parts of water does not coagulate on heating. In experiment 5 
1 does coagulate whereas tube 2 does fiOt* To tube 2 now BLOOD. 
58 
ioc of a 10$ NaCl solution and boil it coagulates at once* 
? * 
Tube 5 contains a fibrinous coagulum whereas tube 4 coagulates 
solid. 
What effect would the addition of NaCl to serum have on the 
coagulating point? 
Compare carefully this and the preceding experiment with exper- 
iment 7 and 8 on albumin. As shown beforeeven slight excdss of aaetic 
acid tends to prevent precipitation of albumin and globulin, 
whereas NaCl favors precipitation. 
4) To 5cc of blood serum in a test-tube add 1 drop of 
formalin, mix and. boil. The blood serum does not coagulate. 
5) To 45cc of water in a small beaker add 5cc of blood serum; 
mix and filter. Receive the filtrate J.n a 50cc graduate, and place 
this in i: beaker of cold water. Pass a current of C0o through 
the diluted serum for about 15 minutes. Then cork ana set 
aside in cold water for some hours. The paragiobulin is thrown 
out of solution as a fine cloud and eventuaTly settles to the bot- 
tom as a white precipitate. 
6) To 50cc of water add lee of blood serum and mix. To 
this dilute blood serum apply the following tests: 
a) . To about lOcc of t. ,e diluted serum add 1-2 drops of strong 
HNO3. mhe clouainass that forms disappears on shaking. 
Now heat the contents of the tube to boiling. A yellowish color 
develops but no coagulation takes place. Divide the liquid into 
two portions. 
(1) Cool one portion then add 5-6 drops of NHO3 and boil. 
Coagulation results. 
(2) Raise the other portion to boiling thon add 5-6 drops of 
NHO3 and boil. Coagulation likewise results. 
b) To 5cc of the diluted serum (1-50) add an equal volume 
of water. This gives a serum diluted 1-100. To this very diluted 
serum add a drop of strong HNO3 and b&il. No coagulation. 
Divide the liquid into two portions. 
(1) Cool one portion then add 5-6 drops of RNO3 and boil. 
(2) . Raise the other portion to boiling then add 5-6 drops of 
HNO3 and boil. The solution remains clear. 
Compare these two experiments and explain the difforence in 
results * 
7) To 5cc of the diluted serum (1-50) add 5-6 drops of strong 
HNQ~. A cloudiness forms and on boUJjig' coagulation takas pl£tce . BLOOD 
59 
a). Repeat this experiment with serum diluted as above under 
c, (I-IDO). Coagulation takes place as in the case of the 1-50 serum. 
8) Boil occ of the dilute serum (1-50) and while boiling hot 
add 5-6 drops of Coagulation results. Compare the volume 
>f the precipitate with that obtained in experiment 6a and 7. 
a). Repeat this experiment with a serum diluted as above under 
b (1-100) only a slight precipitate forms. Qompare the volume 
of the precipitate with that obtained in experiment 6a and 7a. Explain. 
It is evident from the obove experiments that in the heat and 
test for album&n, in the urine or elsewhere, it is nec-essary 
to take into account the amount of HNO3 added and whether the 
solution is cold or h ot. The best result is obtained therefore 
when albumin is present in minute quantities, by adding to the 
cold solution an excess of HNO3 (5-6 drops) to a permanent cloudiness 
and then boiling when a coagulation results. 
HFO3 and heat will coagulate albumin where heat alone will 
fail to do so. This may be the case if the urine tested has an 
alkaline reaction. An additional advantage in the use of HNO3 
is that it will dissolve any phosphates that may be thrown out 
of soMtion by heating the urine. 
9) To 5ec of the diluted serum (1-50) add 1 drop of lX acetic 
acid (lcc of glacial acid diluted to lOOcc of water). A cloudi- 
ness results. epest the reaction of the liquid then boil. 
A coagulum forms and the liquid is perfactly clear. 
a). Repeat this experiment, first raising the dilute serum 
to boiling and adding 1 drop of the \% acetic acid. What is the 
result? 
10) To 5cc of the diluted serum (L~50) add 3-4 drops of dilute...- 
acotic acid used above. Test the reaction of the liquid, then 
boil. No coagulation takes place but the liquid is opalescent. 
a). Boil 5cc of the diluted serum (l-*50), and while boiling 
hot add about 10 drops of lX acetic acid and boil again. The 
cloudiness that forms at first, redissolves. 
In precipitating proteids, from urine or other solutions, 
by means of acetic acid and heht care must therefore be taken 
to add the acetic acid to the cold solution to neutralization and • 
after that to heat to boilingT" 35fven-a—eligWf'acidity cue t 1/r 
acid wril-'L.fte«eprralhupiin in solution. 
Compare the behavior of_acetic-and. hitric acids to the serum 
proteids. 60 
BLOOD* 
11) 3D me of the dilute serum (1-50) add 1-2 drops of HgCl^ 
A white precipitate forms. Shake up thoroughly and divide into 
two portions. 
a) « to one portion add an equal volume of NaCl solution (1-1°) 
The precipitate promptly dissolves. 
b) . To the other portion add an equal volume of undiluted 
serum. The precipitate likewise promptly dissolves. 
The precipitate of mercury and albumin is therefore soluble 
in NaCl, also in excess of proteids. Of what importance is this 
fact in practical disinfection? this test with the similar 
experiment on egg albumin I, 4. Note the difference ftn the behavior 
of the two proteid solutions. 
12) . To some of the dilute serum (1-50) add dilute 
solution till a precipitate forms. Then add a few drops of"strong 
NaOh solution (1-3). The precipitate redissolves yielding a blue 
solution. 
What other substances give similar solutions of cupric hydrate? 
If silver nitrate or lead acetate be added to the dilute serum 
what would be the result? What is the behavior of the salts of 
heavy meatIs to proteids? 
The reaction given by serum ablumin and serum globulin as 
wotked out in the table, will of course be given by the diluted 
blood serum. 
Vo FIBRIN. 
The coagulum obtained by whipping freshly drawn blood is cut 
up into small pieces and washed in running water till perfectly white 
Fibrin on contact with dilute HC1 at 40° swells up and the 
contents of the tube become solid in a few minutes, (See exp. 1, 
peptic digestion). Solution then gradually takes place so that in 
2-3 days the fibrin has disappeared. An acid albumin results,(See 
Exp o 4) . 
Fibrin swells up also in 5)t oxalic acid solution but does 
not dissolve readily. It is also soluble in dilute neutral salt 
solutions. 
Place in each of two test-tubes about 5cc of hydrogen peroxide.' 
To one add a shred of fresh fibrin. Oxygen is eet free especially 
on slight warming through so called catalytic action. This action 
_ is probable due to remnants of leucocytes (nucleotuston) 
To the other tube add some boiled fibrin. What is the result? CHAPTER IX. 
MILK. 
Milk is a secretion of the mammary gland. It is composed of 
water, casein, globulin, albumin, fats, milk-sugar, and inorganic 
salts . The color of milk is due in part 66 the suspended fat 
globules, and in part ho the casein which is held in solution 
by calcium phosphate. The specific gravity of milk from a single 
animal may vary considerably, usually from 1,028 to 1,035, but may 
be as high as 1*039. Market milk which is the mixture of the 
product of .several animals always ranges from 1.029 to 1.034. 
The reaction of milk is usually akkaline or amphoteric. It'amy 
however, be aaid and this is especially true of carnivorous animals* 
On standing milk becomes gradually acid owing to the formation of 
lactic acid by fermentation. Fresh milk does not coagulate on 
heating. After fermentation sets in milk may coagulate on heating* 
and later curdles without the application of heat. Bterlized 
milk, prpperly kept, will remain sweet indefinitely. The scum 
which forms on boiled milk is not coagulated albumin but a com- 
bination of casein and calcium. When removed a new scum forms 
on the milk when heated. Solutions of casein under similar con- 
ditions become covered with scum. 
The addition of rennet to milk produces in a short time a 
solid coagulum, the curd or cheese. The clear liquid remaining 
is the whey or milk-sezuam. The reaction of the nilk is not affected 
by this change. The presence of calcium is necessary to the formation 
of curd. The casein originally present in the milk is apparently 
changed by the ferment into two proteids. One of these unites 
with calcium to form the curd and is known as paracasein. The 
other proteis is formed in small amount, is related to the albu- 
mouses, and is known as whey-proteid. 
Casein is a complex proteid belonging to the nenrheoalbumins* 
It is insoluble in water but readily dissolved in the presence 
<5f alkalis. A solution in calsium hydrate can he neutralized with 
phosphoric acid without precipitation of the casein. fhe milky 
liquid thus obtained contains, in solution or suspension, the 
casein and considerable calcium phosphate. casein is thrown out 
of solution by dilute acids, or by saturation ;with NaCl or MgS04. 
It is also precipitated by metallic salts. In the presence of 
calcium a solution of sasein is coagulated by rennet. As in the 
case of milk, a solution of casein when boiled becomes cohered 
with a scum. On digestion with pepfcin it yields pseudonuclein 
which contains phosphorous. The casein in woman*s milk is dif- 
ferent from that in cow*s milk. The former is more diffficult 
f.O precipitate with acids, salts and rennet. When precipitated 
by an acid the coagulum is finely riocculent and dissolves readXly' 
in an excess of acid whereas casein from cow*s milk is coarsely 
floeculent and is less readily soluble in excess of acid. 
Unlike milk it -does not- on MILK 
62 
digestion. Casein is derived apparently from a nude opr ot eld 
contained- in the protoplasm of the cells of the gland. 
The globulin of mi lie 2 or lactoglobulin of Scbolisn, is pro- 
bably identical with serum globulin. Lactoglobulin is related to 
but not idenfcilal serum albumin. Like casein and milk-sugar 
it is a special product of the cells of the gland. Schlossmann 
found the thr,ee proteids present in milk in the following quantities: 
casein, 3.19%; albumin, 0.38$; *» globulin, Q.15$f« Only traces 
of urea, creatin, etc. arc normally present in milk, consequently 
all the nitrogen present in milk can be considered as contained 
in the proteid substances. 
The fat is present as an emulsion, in the fat globules. These 
vary in size in milk from the same species and from different 
species. According to Woll they are on an average 3,7 U, in di- 
ameter and from 1-to 5.7 million’s of globules are contained in 
one cc of milk. The former belief that the fatty globules were 
surrounded by an albuminous envelope is no longer held. mhe fat 
is supposed to reuult from a degeneration of the protoplasm of the 
cells but it is possible that a part, at least, is brought to the 
gland by the blood. 
The sugar present in the milk, lactose, is a specific product 
of the gland cells and is not directly derived from the blood. 
It is possible that it is derived, like casein, from the nucleo- 
proteids in the cells. mhat these compouhas can give rise to 
carbohydrates has been demonstrated. In exceptional cases milk- 
sugar may appear in the urine. Like glucose it is dextro-rotatory 
and reduces Eehling’s solution. Although readily decomposed 
by bacteria it is not acted upon by pure yeast. This fact as well 
as its solubility, crystalline form and the formation of mucic acid 
on oxidation with nitric acid distinguishes lactose from glucose. 
The colostrum corpuscles can be considered as epithelial cells 
which have taken up fatty globules, rather than as degenerated 
cells. They are found in milk secreted just before and after de- 
livery. And appeqe as nucleated, granular cells containing 
numerous fatty granules. They are from 5 to 25in diameter. 
The milk at this time is yellowish in color, alkaline in reaction, 
and has a high specific gravity 1.046—1.080. When such milk is 
heated it coagulates owing th the presence of increased quantities 
of albumin and globulin. See analysis. 
1) Examine a drop of milk under the microscope. Sketch the 
different sized globules present and measure their diameter. 
They average about but some globules may attain a diameter 
of 18jj&r more. I 
2) . Examine microscopically a drop of skimmed milk. What 
difference is observed between this and Y/hole milk. 
3).Examine with a microscope colostrum milk. Sketch and measure 
the colostrum corpuscles. MILK 
63 
4) Place about lOcc of milk in a test-tube and boil. 
Then immerse litmus paper in the hot milk for 1-2 minutes, remove 
and examine. Under what conditions does milk become acid? 
5) Boil about 25cc of milk in a small beaker for 5 minutes* 
No coagulation proper but a scum may form. Remove the scum with 
a spoon or spatula and ho at again, a new ocum forms. This removal 
of scum will repeatedly take place. What is the nature of the suum? 
Casein is not coagulated by heat. Why does not the albumin in the 
milk coagulate? Save the milk for experiment 13. 
6) To about lOcc of milk in a test-tube add 1 drop of dilute 
acetic acid (L-10), then boil. The casein is coagulated and car- 
ries down with it the fat. ?phe serum is clear. 
7) Set aside in a test-tube some milk over night at ordinary 
room temperature, The next day heat the entente to boiling. 
Explain the result. 
. 8). Place lOcc of milk in each of 5 test-tubes* 
To No. 1 add 1/2 cc cf very dilute HC1 (10 drops of HC1 to 
50cc of water). 
To No. 2 add 1/2 cc of 2% solution. 
To No, 3 add 1/4 cc of saturated (NH4}9C2 solution (1-20) 
Then add to each of these three tubes and also to Nos. 4 and 5 
2 drops of rennet solution and mix. Heat the contents of tube No. 5 
to boiling. Then place all the tubes in a water bath at 40° and 
examine every 3-5 minutes. 
The contents of tube, a will coagulate in a few minutes; 
No. 4 next, Nos. 2, 3, and E will not coagulate. The latter 
does not because the heat has destroyed the ferment. The action 
of rennet is retarded or prevented by the alkalis, and is favored 
by the acids, such as is present in the gastric pice. 
The coagulum which forms contains paracasein and the fat* 
The clear liquid that separates from the coagulum on standing 
is the whey or milk serum. Paracasein is different chemically 
from the casein obtained by the addition of an acid to milk. 
Calcium salts must be present in order that paracasein mpy forrt 
Tube No. 3 does not coagulate because the calcium is thrown out ot 
solution as the oxlate. Compare the change that takes place 
with that in the clotting of blood. If oxlate sodium is added 
to freshly drawn blood what is the result? 
Continue heating tube 3 at 40° for about 1/2 hour. Then add 
2—3 /Irops of CaCl„ solution. The liquid instantly solidifies. 
This shows that the rennet has acted on the casein and chanr*'’ 
Into the modification which, with calcium* yields pararo MILK 
64 
Calcium is likewise necessary to the coagulation of blood, not how- 
ever, for the Srmation of clot directly as in the case of the milk- 
curdo Calcium-free blood plasma (oxlate plasma) and calcium-free 
fibrin ferment when mixed, promptly yield a clot of fibrin* 
The calcium is necessary to the formation of the fibrin ferment 
from a p arent substance, prot hr crab i n (H&mme reton) „ 
9}0 To some milk in a test-tube add 1-2 volumes cf ether, 
close and shake thoroughly* fat globules do not dissolve; 
the milk Remains opaque* Now add a few drops of NaOH and shake 
again. he ether now dissolves the fat and the liquid clears up, 
$his reaction was taken at one time to indicate that the globules 
were surrounded by a albuminous envelop* compare this test wfrith 
the action of ether on blood* 
10) to-some milk in a test-tube add a few drops of NaOH 
and heat. 1T,he liquid becomes yellow, then orange and finally brown. 
11) To a 4% solution of lactose add a little NaOH and test. 
The same color reaction is develpped aas in Exp. 10 which is due to 
the sugar present in the milk. 
12) To some milk add a tincture cf guajac and mix; then pour 
on a layer of old terpentine* A deep blue color develops. 
This test is also given by bd.ood. 
13) Repeat the preceding Exp* using, however, the boiled milk 
from Exp. 5. The color does not develop. Heat has change<T~‘the 
proteids so that they can no longer* assist in the oxidation of the 
guajac resen* 
14) to about lOcc of milk in a tostOtube add 5g of powdered 
and shake thoroughly. Then pour into a filter resting in 
a test-tube and filter over night. Boil the clear filtrate— 
albumin coagulates. tHq casein is precipitated almost completely 
my Mg SO 4* 
15) To 5cc of milk add 4 volumes (fCcc) of strong alcohol 
shake thoroughly and set aside. All the proteids present aire 
precipitated. 
3.6). Dilute lOcc of milk with about 30cc of water and divide 
into three portions. 
To 1 add l-2cc of potassium alum solution (1-10) curd shake. 
The casein is precipitated, and carries down with it the fat. 
To 2 ad cl 1-2 cc of copper s u $ phato- so Ini ion # (1—10) and shake. 
A voluminous greenisn blue pre-clpitate of the proteins present 
jreau Its. M I L K. 
65 
To 3 add about 2 cc. of Alraen* s tannic acid solution and shakec 
The protsids are precipitated. 
17) o Moisten a few granules of pepsin with a drop of water* 
or better with a drop of a 0.7$ NaCi solution. Then add 5 cc, of 
milk, mix and set aside in a water-bath at 40° . Coagulation results 
in a few minutes. It will fal if more water or salt solution is 
added so the pepsin (Pepolharing). Chymesin on digestion with 
pepsin and 0.3$ EC1 is destroyed (Hammersten)• 
18) o Add 50 cc. of milk to about 400 cc. of water, mix well 
and while stirring add dilute acetic acid (1-10), drop by drop, 
till the precipitate becomes coarsely floceulent and ceases to in- 
crease . Stir thoroughly and set aside over night. The reaction 
should be distinctly acid. 
The precipitate consists of casein and fat * Filter off the 
precipitate and allow to drain well, then fold over half the filter 
in the funnel and apply gentle pressure with the fingers until no 
more water can be squeezed out. 
Transfer the precipitate to a small dry beaker add about 30 cc. 
of strong alcohol and stir thoroughly so as to dehydrate the casein. 
Then filter and again squeeze the contents of the filter as dry as 
possible. Transfer the precipitate to a small dry beaker, add 
about 50 cc. of ether and heat Sn a warm water-bath with constant stir- 
ring for about ten minutes. Owing to danger the light should be 
very low or better turned out. Finally transfer the contents 
to a filter and squeeze as dry as possible. 
Spread open the filter on the table, allow the remaining.ether 
to evaporate, tnen powder. The white chalky powder is casein. 
The ether filtrate received in a small beaker on evaporating 
dish siid evaporated cautiously on the water-bath gives the milk-fat. 
The aqueous filtrate from the- casein and fat precipitate con- 
tains albumin and mu lie-sugar. Place it in a. beaker and boil for 
15 minutes* Filter off the precipitate of albumin and reserve 
for subsequent tests. 
Concentrate the filtrate from the albumin in a beaker on a 
wire gauze till it becomes cloudy and bumps. Cool the liquid, the 
cloudiness disappears and is therefore due to phosphates. Heat 
again to boiling and filter hot. Concentrate the filtrate now on 
the water-bath to a syrupy consistency and set aside over night. 
Crystals of milk-sugar separate on standing. 
To the casein obtained in the above, with the biuret apply the 
milion and xanthoproteic reactions. Also dissolve a portion in 
water to which some $T&pCO3 solution has been added.. Observe the 
cloudiness cf the solution. Heat a portion of the casein with 
alkaline lead acetate. What is the result? Apply the same tests 
to specimen of albumin. Review carefully the..reactEons for 3.ac-tosa 
jshd fats. ■ i 66 
CHAPTER X. 
M ILK A N A L Y S. I S 
The milk tc be analyzed should be thoroughly shaken just be- 
fore each portion is taken for analysis in order to insure a true 
sampleo The quantity to be taken is measured out by means od a 
clean and dry lOcc pipette graduated in l/lOcc. The quantity 
taken multiplied by the specific gravity of the milk gives the 
weight of the milk employed for the determination. 
« 
1). SPECIFIC GRAVITY. 
Determine the specific gravity of the specimen by , means -of— 
a) .The picnometer or specific gravity bottle. This is done 
according to directions given. 
b) . The lactometer.--There are two forms of this instrument 
commonly in use. The Suevenne-Muller lactometer, employed largely 
in Europe, gives the specific gravity direct. The lactometer 
of the New York Board of Health reads from 0°, the density of water 
at 15° which corresponds to 1.0, to 120° which represents a specific 
gravity of 1*0348. TOO0 on this scale represents the specific 
gravity of 1.029 which is taken as the minimum density of genuine 
milk. In the absehce of a lactometer the ordinary urinometer 
may be used although the divisions are very small and the reading 
consequantly is not accurate. 
Place a sample of the milk in a suitable cylinder or 50cc 
graduate and determine the density. 
Determine the specific gravity of the skimmed milk obtained 
from the following experiment. What is the result? 
What is the affect of the addition of water to milk? To 
skimmed milk? 
2).Creamomater.—Fill a 50cc graduate to the mark with milk* 
Got aside for 24 hours at the ordinary room temperature. Note the 
volume of the cream and calculate the volume per cent. A good 
milk should give 10-12 per cent of cream. 
Remove the skimmed milk from be&ow the layer of fream by means 
of a pipette and determine the density according to lb* MILK ANALYSIS 
67 
3J. Total solids.—place 2cc of milk in a previously weighed 
watch-glass and evaporate on the water-bath to dryness. Then 
wipe the bottom of the watch-glass and place in an air-bath at 
100-105° for 3 hours. Cool in dessicator and weigh rapidly. 
Calculate the per cent of total solids. This result subtracted 
from 100 gives per cent of water. 
4) Ash.—Place 5cc of milk in a previously weighed porcelain 
crucible, evaporate to dryness on the water-bath. $hen carefully 
ignite so as the char the mass slowly and thus avoid spurting. 
The ignition must be . , continued till the ash is grayish-white, 
free from carbon. Cool in desiccator and weigh. Calculate the 
per cent of ash. 
5) o Pat.■—Roll up in a coil a strio of thick filter paper 
about 2 inches wide and 24 inches long, and tie it with a thresd 
or wire. 5ce of milk is allowed to run slowly on to one end of 
the coil. The coil, dry end down, is placed on. a watch-glass 
and dried in an air-bath at 100-105° for . ..one hour. 
it is then placed in a Loxhlet extraction apparatus which is con- 
nected by means of sound well fitting corks to an inverted conden- 
ser above and to a 150cc wide neck, round flask, cr f!rlenmeyer 
flask below. The weight of the dean dry flask is first ascer- 
tained. By means of a small funnel pour ether into the apparatus 
from above, until it siphons; then add about half as much ether. 
The flask is now heated, cautiously, on a water-bath so that the 
siphon will act about eyeryTive minutes. The extraction will be 
complete in from 1-1 1/2 hours. Then remove the paper coil and 
continue heating till the ether fills the extraction apparatus.and 
is almost ready to siphon. Disconnect the flask and transfer 
the ether from the apparatus to a bottle. The flask still con- 
taons some ether, also water and fat. Heat on the water-bath . 
to dryhess, wipe the flash carefully, and finally dry, in an air- 
bath at 100-105° for one hour. Cool in des.ioa.tor and weigh. 
Calculate per cent, of fat. Subtract this result from that of 
total solids—difference, is Solids not fat. 
In manipulating with ether great cars must be taken to pre- 
vent accidents. The corks must be large enough to fit snugly 
and the gas must be turned off until everything is in readiness to 
begin the extraction. To obtain very accurate results the coil 
of paper should be first extracted with ether to remove what fat 
may be present. 
5). Lactose.—Place about 380cc of water in a beaker, add 
20cc of milk qnd mix thoroughly. Then add gradually about Sec 
of dilute acetic acid,(1—10), with constant stirring, till a floc- 
culent precipitate forms. yhe reaction should be destinctly acid# 
Place Leaker on a wire gause and heat to boiling for 1/2 hour* 
Then filter thonugh a wet filter. Rince the beaker several times 
with hot water, and finally wash the residue on the filter, pro- 
teids and fats, with hot water. Concentrate the combined filtrate 
and wash water to about 150cc. Cool and measure the volume of 
liquid. Determine the lactose-in this solution with Periling* s H 1 LK' A H A L Y S X S 
68 
solution according to the method described* 
lOoc of Fabling*a solution corresponds to Q.C67& of milk sugar. 
Calculate the per cent, of lactose. , 
♦ . / 
7). Casein.-"-To 50cc of water in a small beaker add lOcr of 
milk and mix, Warm on the water-bath to 40°, Then add 2 1/2 
cc of potassium alum delation (1-10) and stir thoroughly < 
finely flocculent precipitate should settle rapidly and the liquid 
should be clear. Let stand for about 15 minutes at 40°, then filter. 
If the filtrate is cloudy pass it again through the filter. Wash 
several times with water* Reserve the combined filtrate and 
wash~water for the next determination. 
Place the filter with its contents in a KJeldahl flask of 
about 250ce capacity, add 15cc , l/4 g powdered CuSQ-t and heat 
on wire gause under the hood* till foaming ceases, then add lOg 
of powdered K„S04 and continue gentle boiling till the liquid is 
light green. * Finally add a little powdered KM11O4, on the point 
of a knife, in order to complete the oxidation and heat till the 
liquid is light grec"• in color. Allow to cool, then transfer the 
contents to a 1 , Miter Erlenmeyer flask* Hince out the digest- 
ing flask.several times with water and add this to the acid solution 
Dilute the contents of the flask to aboist 6G0cc and cool* Add 
a little powdered talc on the end of a knife* Insert in the neck 
of the flask a double perforated stopper rubber, provided with a 
Eeitmalar bulb and a thistle tube. The end of the latter should 
roach nearly to the bottom of the flask. A long strip' of red litmus 
paper should be suspended from the nock of the flask and should 
extend down into the liquid. Oonnect the free end of the Reitmaier 
bulb with a condenser. The lower end of the condenser is con- 
nected with a bent tube which extends down into the liquid of the 
receiving flask. This flask should be about 250cc capacity 
and contains £0cc of n/lO oxalic acid, which will unite with the 
NH* that will be distilled off a When all is in readiness pour 
into the flask, though the tube, strong NaOH solution (1-2; until 
the liquid is decidedly alkaline. About 60-60 cc. will be required 
Heat the large flask and distil over about POO cc* Then replace 
the receiver by a flask containing 10 cc. of n/10 oxalic acid and 
f5oma water and continue the distillation till about 100 cc. of 
distillate passesover. 
To each of the receivers now add a few drops of alcoholic 
rosolic acid and titrate with n/10 NaOH to a strong pink reaction. 
The second flask serves as a check and should be free, or nearly 
free from ammonia. The difference between the number of cc. 
of oxalic acid employed and the number of cc. of n/10 NaOH necessary 
to neutralize the distillate gives the number of cc. of n/10 NHg 
given off in the distillation. 
A blank experiment with filter paper, 15 cc. of H2SO4 and salts 
- as above should be carried through in exactly the same manner to 
ascertain how much may be given off by the .reagents themselves. 
The number cf cc. of ft/10 NH3 thus found should ba subtracted as a M IKK ANALYSIS 
69 
correction from the total number of cc, of n/10 Nil given off in 
the above» The difference multiplied by the n/10 factor of nitrogen 
0.0014, gives the amount of nitrogen contained in the casein pre- 
cipitate from 10 cc* of milk* This amount of nitrogen multiplied 
by 6o37 gives the amount of casein in 10 cc, of milk. Calculate 
the per cent* of casein* 
8) GLOBULIN AND ALBUMIN* — 
The filtrate from the alum precipitate of casein in the 
proceeding experiment contains globulin and albumin* To this" fil- 
trate add 10 cc, of Almen's tannic acid solution. Filter off the 
voluminous precipitate of proteids, wash -several timeswith water, 
and allow to drain. Place the filter arid contents in a Kjeldahl 
flask and determine the ni trogen as above, making the proper ccrree 
tion for blank. The amount of nitrogen found multiplied by the 
factor 6,37 gr'ves the amount of albumin and globulin in 10 cc* of 
milk, Calculate the per cent, of albumin and globulin. 
Amen’s tannic acid solution is prepared according to the follow- 
ing formula: Tannic acid 4 g,; 8 cc of 26% acetic acid; 90 cc* 
of 9G/o alcohol; 100 cc. of water. 
9) TOTAL NITROGEN IN MILK.— 
Place 10 cc. of milk in a Kjeldahl flask add 15 cc. of 
and treat as above under exp. 7, This gives the total 
gen present in the milk and serves as a control on the two proceeding 
de t e rminations. 
A report of the results obtained is to ba made out, as follows; 
together with a statement as to whether the milk is adulterated or 
not: 
1. Specific gravity, whole milk . . . * . 
1. Specific gravity, whole milk, ...... 
2. Specific gravity, skimmed milk, . . . . * 
. are am 3 per , oi, . . * . . . « . <> »» 
4. Water, n w R .......... 
5. Total solids K 
6. Total solids not fat, per cent of, . . 
7. Fat, per cent of, ............ 
8. Ashy n ” " ........... 
9a Lactose, n T! 
10. Case in ** • ••••••♦•••* 
11. Globulin and Albumin, per cent of, . . . 
12. Total nitrogen as proteid, per cent of. . 
To decide upon the purity of a milk the determination given 
under (4), 1, (4), 5, (6), 7, are as a rule sufficient* In case 
of doubt the ash may be determined. The legal standard of milk 
varies in different states. In Now York the minimum of total 
solids allowed is 12/?; of fat 5 fL In Massachusetts the total 
solids must not fall below 13;?. New Jersey allows a minimum of 
12% for total solids. MILK ANALYSIS. 
70 
The following table, compiled froxrt'Konig, shows the average 
percentage it ion of various milk: 
Milk of, 
i 
Ho. of 
analyses 
averaged 
Specific 
Grav ity 
Water 
Casein 
Altunin *Fat 
Lactose: Ash.; 
* * 
# • 
« • 
Woman 
107 
1.027 
87.41 
1.03 
L.26 :3.7S 
6.21 
0.31: 
Cow 
793 
1.0315 
87.17 
3.02 
0.53 :3,69 
4,88 
0.7lj 
Cow> 
(Colostra 
Tu) 42 
— 
74.67 
4 a 04 
13,60 :3o 59 
2.67 
1.56 
Goat 
38 
— 
05,71 
3 o 20 
1,09 :4.78 
4.46 
0.76 
Sheep 
32 
1.034 
80.82 
4.79 
1,55 *6.86 
4.91 
0,89 
Mare 
47 
1.0347 
90.78 
1 o 24 
0.75 .1.21 
5.67 
0.35 
Ass 
4 
89 0 64 
0.67 
1.55 *1.64 
5,99 
0,51 
Hog 
7 
— 
84.04 
'VS '.4,5 5 
3 * 13 
lo05 
Bog 
28 
■ 75.44 
6.10 
i 5 o05 :9.57 
3.09 
0.73 
THE END appbukix. 
Ehrlich5s Diazo-reaction. 
The reagent employed for this reaction should be freshly 
Two solutions are first prepared, 
(Ij— To 1000 C.Co of water add 50 C.C. of K 01 and 5 g. of Sul- 
£Mnilic Acid, 
(2)-—A 00 5;b solu tion of Sodium Ni tri te* 
Just before use these two solutions can be mixed to form the 
reagent proper as follows: To 250 C,0o of solution lio.l add. 5 C.C. of 
solution IIo, 2. Or, on a smaller scale, to 5 CMC. of Ho, 1 add 3/4 
drops of solution No, 2, The Nitrite solution is subject to oxidation 
on standing, and should not therefore be prepared in large quantity* 
Mix the urine with an equal volume of the reagent, and add 
at once an excess of II H4O II . A pink to a deep re-7 color, an-1 es- 
pecially a pink colored foam, constitutes the diazo—reaction. 
Normal urine as a rule gives a brownish yellow, very rarely a pink- 
ish color. The reaction is very rare in Chronic non-febrile diseas- 
es. It is met with as a rule, excepting in very light cases, in ty- 
phoid fever, and a certain diagnostic value is therefore ascribed to this 
reaction. It has been found however, in exant hemic typhus, in small- 
pox, in acute initiary tuberculosis, in severe tuberculosis, and. in 
pneumonia. The dissapearance of the reaction in typhoid urine may 
| be taken as a favorable sign, while the appearance of the reaction in 
tuberculosis is an unfavorable sign. 
The substance which gives this reaction is unknown. It is an 
aromatic compound, probably a metabolic product which appears in the 
urine only under certain special conditions. 
The reaction resembles somewhatnthe test for nitrites as given in 
experiment 11 under Saliva. If nephthylamine is replaced by AyNapli- 
the i the reaction is even more similar. 
A P P E II D I X I I * 
Estimation of Alloxuric Bodies and Bases* 
Uric acid and the nuclein bases are known to possess an 
alloxan group and an urea group in their molecule. They are more-* 
all precipitated on boiling with a solution of Copper Sulphate and 
a reducing agent. The term Alloxuric bodies lias been given to all 
those constituents of urine which contain the two groups mentis 
deducting from the Alloxuric bodies the uric acid present in the urine* 
'leaver the alloxuric bases. The latter therefore includes the nu- 
clei ii bases* as well as other related compounds which have not PS yet 
been isolated from the urine. 
The reagents employed are a 13/ solution of Copper Sulphate; 
a solution of HOvtiuei acid SuplMte Jl; -an! lo/ aolu.tim of .Barium 
Chloride. The 131 hod is as follows: APPEUDIX 
72 
Place 100 C.C. of the Albumin-free urine in a bearer nnd 
boil, then add 10 G.C. of the Copper Sulphate solution and 10 C»C, of 
the Sodium acid Sulphite solution and boil. Later add 5 C.C. of the 
Barium Chloride solution in ord.er to cause the precipitate to settle 
more readily. Let stand tv/o hours. Then transfer to a snail pie it* 
ed filter and wash five times with water heated to 00c. Then plao* 
the filter and contents in a KieldaiBL flash, cirri determine the Hi trojan 
present ac cor dire to hieldahl’s method as given uehr Mil]:. 
A blank experiment must be made, using a clean filter paper, in- 
stead of the one with the precipitate. The number of C.C. of --eci- 
normal Ammonia which is found, in this blank experiment must be •1e<kiot- 
e 1 from the number of C.C. found, in the above experiment. The dif- 
ference represents the number of C.C. c-f deeinormal rtunonia formed from 
the Ailoxuric bodies in 100 C.C. of she urine. Therefore, this num- 
ber of C.C. multiplied bp the decinormal factor of nitrogen gives the 
Hitrogen in the Ailoxuric bodies. 
In another sample of the urine determine the amountof the uric 
acid according to the Salkowski-Ludwig method? as given in the book. 
Calculate the amount of the uric acid as contained, in IOC C.C. of the 
urine, an-1 then the amount of ITitrogencontained in the uric acid pres- 
ent in 100 C.C. of urine. 
Subtracting the found nitrogen of uric acid from the found nitro- 
gen of Ailoxuric bodies gives the ITitrogen, of Ailoxuric Bases. 
The ratio of the Pi trogen of ailoxuric base§ to the nitrogen of 
uric acid is about 1—4 in normal urine. In laukamia it may rise to 
1—1, 
Do term!nation Of Iota 1 ITi trogen Tc Urine. 
Place 5 C.C. of urine in a k.ieldahl flash an-1, determine nitro- 
gen present ac cor-Ting to hi eldahl * s method. Receive the -list! Hate in 
50 C.C. of decinormal oxalic acid. 
a p ? e :•! d i :: in. 
Qua n ti tatjnres,Analysis of Gas tunic Ju ice. 
TohPTbR1 S IUuTJIOD^—"—^The following reagents are necessary; 
(1) —Decinoraal Sodlun hydra t e solu tion. 
(2) —A X' Alcoholic solution of Phenol-pfcthaloin. T'uio indicates 
total aci Is. 
(3) —A 1/ ' a qu sous s o lu t i o n of So linn olizainsulphonie a c i <1 • 
This indicates all a 0 i 1 except the loosely eonline!. 
(4) —A 0.5,2' Alcoholic di-methyl andJoazobenzol solution* This 
indicates only free MCI. 
The method, is as follows: Measure out into 3 beakers 
10 C.C. Of the filtered Gastrri-c Juice. If necessary a smaller amount 
may ho taken, or the Gastric Juice nay ba. diluted. APPENDIX 
73 
To beaker ho. 1 add 1-—.? "Trops phenol phthalcin, then run in *eei~ 
normal Sodium Hydrate, not to the first pink color, but to a dark red <. 
whioh no longer increases in depth. Note the number of C.C. of re- 
adout employed. 
To beaker lie. 2 add 3—4 drops of the Alizarin solution and then 
titrate with dec!normal Sodium Hydrate until the first pure violet, 
color is reached. note the number of C.C. employed. 
To beaker No. 3 add 3—4 drops of di-methyl amidoazobenzol solu- 
tion. A yellow color indicates the absence of free II Cl. If a red 
color is present run in dec!normal Sodium Hydrate till it just dissap- 
pears. note the number of C.C. employed. 
The difference between the number of C.C. of decinum&'X Sodium 
Hydrate required for beaker ITo. 1 (total acids) and the numb ;r of C.C. 
required for beaker No. 2 (all acids except loosely comb hie-'}, gives 
the loosely combined II Cl. 
The number of C.C* require*1 for beaker No. 2 gives the free II Cl. 
The number of C.C. require*! for beaker No. 1 gives the total Acid-* 
i t y. 
The number of C.C. of reagent required for beaker ITo. 1 minus the 
number of C.C. required for beaker No. 2, minus the number of C.C. 
found to correspond to the loosely combined II Cl. gives the Organic 
Acids and Acid Salts. 
APPENDIX IV. 
The Mitscherlich Polar!meter. 
The instrument consists of two IJicol prisms, the polarizer 
and analyzer, enclosed in brass tubes and supported in such a way that 
they can be rotated; the tube containing tue analyzer nas a pointei 
attached which measures the amount cf its rotation upon a circle 
graduated in degrees. Between the two Hicols is placed the observ- 
ation tube, a brass tube exactly 200 H.M* long, the ends of wnich ai e 
closed with glass plates; this holds the solution to be tested.. 
Adjust the instrument as follows: 
Place a lamp behind the polarizer and fill the observation 
tube with distilled water; set the pointer at 0°, and then rotate the 
polarizer until the field becomes darkest. The polarizer must nob 
be moved again. 
As the instrument now stands, the two Nicols have their section 
at right angles. If the analyser is rotated, one way or the other, 
the field gradually becomes brighter and is brightest when the pointer 
is at 00°i the sections of the prisms now being parallel. The field 
will be dark again at 180°. 
Starting With the instrument adjusted as above, fill the observ- 
with the solution to be tested.. If this has the- power-o£ A P P E lf D I Z 
74 
rotating the plane of polarization, the field appears brierht; The 
analyzer must now be turned to the right or left till the field again 
becomes darkest, thus compensating for the rotatory power of the solu- 
tion,. This shows whether the substance is Textrc or lawo—rr>tai3grry. 
By knowing the length of the tube, the eoncentration of the solution,, 
and the number of degrees through which the analyzer was turned, the 
Specific Rotatory Power can be calculated^ 
a p p e n d i 
o 1 e t xjte <&ac c hartae to r i 
Tills instrument is use-I only for the purpose of determining 
the percentage .of cane sugar in a given sample. 
It consists of two Hi col prisms, the analyzer and polarizer, and 
the observation tube placed between then*. Between the polarizer 
and source of light is the regulator, a Nic'ol prism and a quartz plate, 
for the purpose of changing the colors. Between the analyzer and. 
tube is the compensator: this consists of two wedge-shaped plates, one 
fixed, and the other capable of being slid over it, thus increasing or - 
diminishing the thickness of the crystal through which the polarized 
ray passes. Fastened to the movable plate is a scale graduated so 
that it can 'be read to tenths of one percent;, the reading is dene by 
means of a vernier and telescope.- The source of light is a lamp 
placed back of the polarizer. 
171 th the scale reading at 0°, and the tube filled with dis- 
tilled water, the field appears as a colored circle divide! vertical- 
ly, and. both halves of exactly the same shade of color.. This color 
may be changed by simply rotating the regulator*, For most persons 
the "sensitive tint" is a rose violet. 
Now fill the tube with, a solution of cane sugar, prepared 
as given below* The plane of polarization is deviated, and the two 
disks are of different colors. Then turn the screw of the compens- 
ator till the disks are again of the same shade, thus compensating* for 
the deviating effect of the sugar. The percentage of cane sugar can 
now be read, distinctly from the scale. 
The instrument is so made that with a solution of pure-cane 
20*043 gras, in 100 C.C. at 17.5° C* the reading will 
be loo;/. Consequently, in making a detemination, dissolve 26, 43 
grma*~~of the substance in distilled water at 17*5°, and dilute te 100 
h+C, The reading obtained will be the percentage oare^agajr^iTt 75 
APPENDIX VI. 
“pancreas . 
The pancreatic secretion is a clear thick alkaline fluid, rich in 
solids and possess very active ferment properties. It contains at least 
three distinct frnients besides albumen, leucin, fats, soap, and salts. 
These solid constitients make up about 10% of the secretion. After a 
pancreatic fistula has been in place for sometime the secretion is 
altered. It becomes thiner, strongly alkaline and shows little or no 
proteolytic action. The amount of solids in this altered secretion 
scarcely exceeds 2 per cent. The quantity of the secretion given off 
in a period of 24 hours is not definitely known. 
The ingestion of food stimulates the flow of the gastric juice.. 
There is, therefore, no secretion during starvation and in carniverous 
animals where some time elapses between meals it is intermittent. Cn 
the other hand the secretion is going on almost continually in herbiv- 
orous animals because digestion is uninterruptedly taking place. 
As stated above the pancreatic secretion contains at least three 
distinct ferments or enzymes splitting up respectively fats, carbohy- 
drates and proteids. 
The neutral fat which is taken into the body with the food is 
acted upon by one of the ferments steapsin or pialyn and is split up by 
hydration saponification into free fatty acids and glycerin. This 
ferment is very readily decomposed by acids and may be absent therefore 
from old pancreas. Only a small portion of the fat, however, undergoes 
this change. The free acids now combine with sodium carbonate to form 
soaps and the resulting soap solution readily emusifies the 
remaining neutral fat and thus brings it into a finely divided jon- 
dition suitable for absorption. A considerable portion of the fat , 
at times, be decomposed into free fatty acids through the activity of 
bacteria. The free fatty acids are not absorbed as such, but appear 
to be regenerated in the intestinal walls, by synthein, into neutral 
fat. Only a very small amount of fat seems to be absorbed as soap: 
The cleavage of fats by the pancreatic ferment and the subsequent 
emulsification is necessary to the proper absorption of fats. In ad- 
dition to the pancreatic secretion, the bile plays and important part 
in the absorption of fat. It is well known that closure of the bile-, 
duct, whether experimentally, or in disease as in icturus, is followed 
by diminished absorption of fat and increased excretion of fat, more 
especially fatty acids, in the feces. Some part, however, continues 
to be absorbed even in the absence of the bile secretion. 
The pancreatic secretion, however, is necessary since no absorption of 
fat takes place when the pancreas is extirpated. In the latter case, 
however, milk continues to be absorbed owing to the already emulsified 
condition of the fat. Some fat may,at times , be absorbed even after 
total extirpation of the pancreas since bacterial ferments may split 
up the fat and thus emulsification and hence absorption may result. 
The second ferment of the pancreas acts on starches splitting up 
the bodies into dextrin and iso-maltose. This ferment is spoken of as 
amylclytic or diastatic and resembles in its action the ptyalin of the 
saliva. It le probably not identical with, the saliva ferment. It is A P t E ft. 1> I X. 
76 
solu&ble in water, and in glycerin; insoluable in alcohol. This 
diaatatic ferment appears to be absent during the first few weeJCe of 
infant Mfe. At the temperature of the body it acts rapidly go boiled 
etarfih.- ‘ converting this into amylodextrin, erythrodextrin, achfOodffc*- 
trin, iso-maltose and maltose. By the action of a special inverting 
ferment the maltose then is converted into glucose in which form the 
carbohydrates are chiefly absorbed. Other mono-saccharides as Iaevu~ 
lose and galactose may also be absorbed direct. It is possible for 
small amounts of dextrin and for milk sugar to reach absorption. 
Sugar is absorbed very rapidly so much so, indeed, that if a very 
large amount be ingested at one time it appears in the urine. This cor 
ditionknown as alimentary glycosuria, does not accur when large 
quantities of starch are ingested. Although the pancreatic gland is 
iiecessary to the complete absorption of all the starch ingested, it is 
a note-worthy fact that about one-half of the starch ingested will 
still be absorbed after total extirpation of the pancreatic gland. 
This may be explained by the diastatic action possessed by many bacter- 
ia. 
The third ferment of the pancreatic secretion is proteolytic in 
its action and is known as trypsin. This ferment does not exist as 
such in the substance of the gland but is represented by a parent-sub- 
stance trypsinogen which is most abundant in the gland in from 14-18 
hours after a meal. This zymogin during the process of secretion is 
converted into enzyme trypsin. Just how this takes place is not 
definitely known. This conversion con be accomplished artificially 
by the action of air, water, acids, very weak alkalis ana various other 
substances. It is probable that, as in the case of pepsin, the pan- 
creatic secretion of different animals contains slightly different 
trypsins. Stronger alkalis present the cleavage of the zymogen. 
Trypsin, like other in its purest condition proteid re- 
actions. It is soluable in wat er, insoluable in alcohol and glycerin. 
When in an impure state, however, it may be dissolved by glycerin. 
This is true of the Other enzymes. In neutral or slightly alkaline 
solution it is readily destroyed at 50 degrees. It is also destroyed 
by gastric juice and unlike pepsin it digests fibrin in alkaline, 
neutral or even very fainly acid solutions. It is destroyed by 
mineral acids but not as a rule by organic acids. The fibrin in 
tryptic digestion does not swell and is not irregularly eaten away as 
is the case in peptic digestion. The fibrin digestion with 
trypsin takes place most rapidly at about 40 degrees and in slightly 
alkaline solution ( 0.3/* NagCo™ ) . 
In view of the fact that trypsin acts best under the conditions 
mentioned, it is evident that the products of the tryptic digestion 
will be mixed with various bacterial products unless special attention 
is given toward inhibiting the growth of these micro-organisms♦ 
In. *ctpal experiments therefore thymol or Chloroform is added to 
.suppress t&e bacteria. In the intestines, of course, during 
ic digestion the bacteria are unhindered in teir action. 
Among the products resulting from the action Of trypsin proper, , . 
on fibrin may be mentioned albumoses, pepton, leucin, tyrosin, aspara- 
giniG acid, lysin, ammonia and proteinochromogen. True pepton is 
formed much more readily in, tryptic than in peptic digestion. This 
pepton is eventually * of the kind known as antipepton, whereas 
the hemipepton has been decomposed yielding products such as leucin, 
tryosin etc. Trypsin dissolves gelatih yielding a gelatin^pwpton. 
The collagens or gelatin—yiolding -connective tissues are not acted 
upon until they have been altered by haat or acids. Trypsin has no 
action on fats or carbohydrates, 
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