PETERS-HILTNER,

SF
NJ R53
S 9m

CORNELL UNIVERSITY.

THE

Roswell PP. Flower Library
THE GIFT OF

ROSWELL P. FLOWER
FOR THE USE OF
THE N. Y. STATE VETERINARY COLLEGE.
1897

Cornell University Libra:

ethods for the examination of milk; for

DATE DUE

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Library

The original of this book is in
the Cornell University Library.

There are no known copyright restrictions in
the United States on the use of the text.

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Ae sper

METHODS

FOR THE

EXAMINATION OF MILK

FOR
CHEMISTS, PHYSICIANS AND HYGIENISTS,

COMPILED BY

DR. PAUL SOMMERFELD,

ASSISTANT IN CHEMISTRY IN THE EMPEROR AND EMPRESS FREDERICK HOsPITAL
FOR CHILDREN OF BERLIN.

WitH A PREFACE

BY

DR. ADOLF BAGINSKY,

PROFESSOR OF MEDICINE AND SPECIALIST IN DISEASES OF CHILDREN, IN THE
UNIVERSITY; DIRECTOR OF THE EMPEROR AND EMPRESS FREDERICK
HOSPITAL FOR CHILDREN OF BERLIN.

TRANSLATED BY

DR. A. T. PETERS,
INVESTIGATOR OF ANIMAL DISEASES, UNITED STATES EXPERIMENY STATION
OF THE UNIVERSITY OF NEBRASKA.

AND

R. S. HILTNER, A. M.,

INSTRUCTOR IN CHEMISTRY, UNIVERSITY OF NEBRASKA.

CHICAGO:

ALEX. EGER,
1901.

i
Copyrighted
at Washington, D. C.,
by
ALEXANDER EGER,
1900.

TABLE OF CONTENTS.

CONTENTS «c.ceeiccnce ta suhearangmas mamangnaei aad iawls §
TRANSLATOR’S PREFACE ...............000045 9
INTRODUCTION. since paanvcuae ngs Weapenen saunas dH,
I. QUALITATIVE EXAMINATION OF
MILK.
EXAMINATION OF THE FATS................ 13
AL BUMINOMLD SS wsiscccassuenonnalacn ania 24 bmhineins aiaareeus 17
MILK SUGAR cxcusuiies omnanguuennumiaadee eee 18

DETERMINATION OF SPECIFIC GRAVITY...21
II. METHODS OF QUANTITATIVE ANALY-

SIS.
THE QUEVENNE LACTODENSIMETER
(WHOLE: MIL jeden ones cane ucesemace see eng
THE QUEVENNE LACTODENSIMETE
(SKIMMED MILR) ..............00.........- 24
DETERMINATION OF ACIDITY...............25
DETERMINATION OF TOTAL SOLIDS........28
DETERMINATION OF ASH..................... 29
DETERMINATION OF FATS................04.,. 31
SOXHLET’S GRAVIMETRIC METHOD OF
PSN ASIST Sos sadsculisiatadsacips duianuuadayn up eaeereiaands 31
EXTRACTION OF FATS BY MEANS OF PE-
STROLEUM BLA ERS wiccususadecewas vegwewed 33
SOXHLET’S AEROMETRIC METHOD......... 35
METHODS WHICH GIVE ONLY APPROXI-
MATELY ACCURATE RESULTS........... 38

DETERMINATION OF TOTAL NITROGEN... .42
METHODS OF PRECIPITATION OF ALBU-

ATEN OTD SY 6 s.canicas,emaeturicotrereannan nena g ahaa 46
PRECIPITATION WITH TANNIN... .0..00440- 47
THE RITTENHOUSE METHOD \uucevscesceeds 48

5

6 CONTENTS.

PRECIPITATION WITH TRIOHLORACETIC

AGLD \ 254 toes hosts 14 Shee Ay esate ee Ree 49
TITRATION WITH FEHLING’S SOLUTION. ..50
DETERMINATION OF LACTOSE.............-- 50
SOXHLET’S AND ALLIHN’S GRAVIMETRIC

METHODS » covacteeg varcisev ant area eoteane. 52
ESTIMATION BY MEANS OF CIRCULAR PO-

LARTZATION sosisangiete peg tamirineah Aste ee eo 55
DETERMINATION OF CITRIC ACID........... 56

lI. DETECTION OF PRESERVATIVES.
BORAX AND BORIC ACID..............-00005. 60
BORIC ACID, SODIUM CARBONATE, BICAR-

BQ INCASE Bis icacacesvae ovarian se eabsauscbeavaud a beddoe avarccendiareumete 61
SALICYLIC ACID AND BENZOIC ACID........ 62
DETECTION OF BENZOIC ACID............... 62
DETECTION OF FORMALDEHYDE............64
CHROMIC ACID, BICHROMATES, AND MER-

CURY SALTS BUC. ices sewed ac aasteaciees oie 65

SODIUM FLUORIDE AS A PRESERVATIVE. ..66
IV. DETECTION OF ADULTERATIONS.

ADULTERATION WITH WATER............... 67
ADULTERATION WITH STARCH, DEXTRIN,
ELC. scsi gandowes cand eae oP ewgdewee Eee es naelaes 71

V. ESTIMATION OF INSOLUBLE FOR-
EIGN MATTER.

DETERMINATION OF INSOLUBLE MATTER.72

VI. EXAMINATION OF CONDENSED
MILK AND CURDLED MILK.

RENCK’S METHOD

CONDENSED MILK......................00.005. 75
CURDLED MIE vsnenoi eta me ve quien saan ak GOs oekoe 75
METHODS OF PREPARATION AND CULTI-
VATION ssa wssemies as tagedal rag asennad date 77
QUANTITATIVE ESTIMATION OF GERMS:
MILK ANOMALIES .............00.0..0.0000% 78

DEMONSTRATION OF INDIVIDUAL PATH-
OGENIC BACTERIA IN MILK.............. 84

CONTENTS. 7

DEMONSTRATION OF BACILLUS TUBERCU-
LOSS: nad axciegunighaaeroandecin Nema dacanly eis aaae 84

LUS

VII. APPENDIX.
EXAMINATION OF POWDERED, MALTED
MILK AND OTHER SOLID FORMS OF

PRESERVED MILK ansyssemetack supe eeass RG
VII. BIBLIOGRAPHY.
BACTERIOLOGY OF MILK. vA
PRESERVATIVES AND ADULTERATIONS.
METHODS OF DETECTION. hacwets as arnsaiinns O3
VARIATION IN THE COMPOSITION OF
MILE, ccnmeenrxemoomdeivercsbsdederscsannerte 93
DETERMINATION OF THAIS is acunisterescie yosevonn aie en OF
COMP OSERION, scacininsaiaiur Hiatal navies -esgignraetaaas OS

METHODS. OF ANALYSIS: ::cccexenevise osane. 28

PREFACE.

The necessity of providing nurses in children’s hos-
pitals with a good article of food in a pure, unadulter-
ated and convenient form for their patients, has led The
Emperor and Empress Frederick Hospital for Children
to pay special attention to the fulfillment of this need.
Definite arrangements were made with an owner of a
dairy in close proximity for controlling the selection of
cattle, method of feeding, care and miiking, thereby se-

curing a good clean product.
The prompt fulfillment of these terms was under the

control of Dr. Sommerfield, and it is through his efforts
in ascertaining the most expedient and efficient methods
ot control that this little book has been published.

It will certainly be of interest to those who are en-
trusted with the care of children in similar institutions
to learn of this publication, and we hope, also, that it will
be of equal service to many others.

ADOLF BAGINSKY.

TRANSLATORS’ PREFACE.

The general excellence of this little work justify the
translators in presenting it in this form, with the permis-
sion of the author, to the English-reading public. The
literature on the subject of milk analysis is by no means
limited, but there is a noticeable paucity of manuals or
guides adapted to the needs of commercial analysts, mar-
ket inspectors and health officers.

The translators have adhered as closely as possible to
the original text. They have taken the liberty to make
a few additions in the way of foot-notes. In cases where
the methods given do not accord in detail with those
commonly practiced in this country at present, refer-
ences are made to the standard literature on the subject.
A short bibliography has been added in which is cata-
logued some of the recent researches along lines within
the scope of this work.

DR. wl Ts PETERS,
R. S. HILTNER:

INTRODUCTION.

Milk, which is a secretion of the lacteals of all female
Mammalia, is especially characterized by the presence of
three elements common to it: casein, butter fat and milk
sugar. Besides these there have been found a large num-
ber of substances of more or less importance in different
kinds of milk.

The mineral substances, phosphoric acid and calcium
salts are of importance in aiding the digestibility of milk.

In close relation to the above is the ever present citric
acid in cow’s milk. In normal milk there was found
among other substances, alcohol, acetic acid (Bechamp),
ammonia (Latschenberger), milk sugar, urinous sub-
stances (Bouchardat, Quevenne, Morin, Picard, Lefort),
lecithin, cholesterin, hypoxanthin (Tolmatcheff, Schmidt,
Muhlheim), flourine (Wilson), sulpho-cyanic acid (Mus-
so), and kreatinin (Weyl).

The milk of different species is distinguished not only
by its physical characteristics and distinct quantities of
substances present, but also by the fact that they show
themselves different toward various chemical reactions.
For instance, the casein of human milk is a different sub-
stance from the casein of cow's milk (Biedertfi, Camerer,
Lehman). The fat of various kinds of milk presents dif-
ferent chemical reactions, whereas the milk sugar in all
kinds is the same (Deniges).

Examination of milk for estimating its value, embraces
the quantitative determination of the specific gravity, dry
matter, albumin, fat, sugar, ash, phosphoric acid in the
ash, and dirt, and the detection of adulterations and pre-
servatives.

11

QUALITATIVE EXAMINATION OF MILK.

The analyst is seldom called upon to decide whether
the fluid under examination is really milk. If such a
case should arise, the essential constituents of the ma-
terial, namely, fat, casein, and milk sugar, should be isy-
lated,

I. FATS.

The fat may be extracted by one of the following
methods. By noting the appearance and properties of the
butter fat, an index may be had to the quality and source
of the milk in question. The ether solution of cows’
butter has a faint yellow color; that of human colostrum
has an orange or ruby-red color (Pfeiffer), while that
of goats’ milk is colorless (Schaffer). The milky tur-
bidity of the fluid does not disappear by shaking with
ether.

The exact composition of the butter fat in question
may be obtained by determining the volatile fatty-acids
according to Reichert’s method, and the insoluble fatty-
acids by Hehner’s method. The latter is based on the
fact that butter has about 87 per cent. of water-insoluble
fatty-acids (varying between narrow limits according to
the time of year and the kind of food), while other animal
and plant fats contain from 92 per cent. to 95 per cent.
of these acids. The analysis, which requires about 200
cc. of milk, is carried out as follows:

Of the previously weighed sample of butter, take
three or four grams by means of a glass rod and place

1S.

14 QUALITATIVE EXAMINATION OF MILK.

it in an evaporating dish 1o to 12 cm. in diameter.
The glass rod with the adhering fat is placed in the dish.
The butter sample is again weighed, and in this way the
exact amount of butter-fat taken may be found. One
now adds to the butter-fat 50 c. c. of alcohol and one to
two grams of pure potassium hydrate. The whole is
gradually warmed on a water bath, when the butter-fat,
especially when stirred, quickly dissolves to a clear, yel-
low fluid, and a strong odor of butyric acid ester be-
comes noticeable. The heating is continued for about
five minutes, and distilled water is then added, drop by
drop. Ifa cloudiness due to undecomposed fat be no-
ticed, continue to warm it until at last the addition of
water to the fluid does not produce the least turbidity.
The saponification is then completed. If, through care-
less addition of water, the fat separates out on the surface
as oily drops which will not dissolve in the diluted alco-
hol, it must be evaporated to dryness and then redis-
solved in alcohol. In such cases it may be better to com-
mence the process again with fresh material. If the
diluting with water is carefully done, it is very rare that
a separation of oil occurs.

The clear soap solution is put on the water bath to
drive off the alcohol and evaporated to a consistency of
syrup. The dish is then removed from the bath and the
contents dissolved in 100 to 150 c. c. of water. To de-
compose the soap, add to this clear fluid a slight excess
of dilute sulphuric acid or hydrochloric acid. In this
way the insoluble fatty-acids are precipitated out as a
curdy mass, which to a great extent rises rapidly to the
surface. The material is heated for about half an hour,
until the acids melt to a clear oil and until the watery
fluid beneath becomes perfectly clear.

In the meantime, select a very thick Swedish filter,
to cm. diameter, and dry it in the water oven. The filter

EXAMINATION OF THE FATS. 15

paper must be of the best quality and so dense that even
hot water will only percolate through it drop by drop.
Weigh a small beaker, a funnel, and also the funnel plus
the filter. In this way one may get the weight of the
filter plus the beaker. (The hardened filters made by
Schleicher & Schull are especially adapted to this work.)

The weighed filter is pressed tightly into the funnel
and thoroughly moistened and half filled with water.
Then pour out of the evaporator the watery fluid and the
molten fat, and wash the dish and glass rod with boiling
water. It is not difficult to transfer all of the oily mass
to the filter, so that there need not be a trace remaining
on the dish. To be certain, one may wash the dish with
ether and afterwards add the material thus recovered to
the insoluble acids. As a rule the amount recovered
from the dish with ether in this way is very small, usu-
ally less than one milligram.

The fatty-acids are now very carefully washed on the
filler with boiling water. Never fill the funnel more
than two-thirds full. When the filtrate tested with sensi-
tive litmus paper no longer reacts acid (three grams of
fat usually require three-fourths of a litre of boiling
water), allow the water to drain out and then dip the
funnel and contents into a beaker filled with cold water
so that the surface of the water is the same within as
“jnoYIM As soon as the contents of the filter becomes
solidified, it is removed from the funnel and placed in the
weighed beaker and dried to constant weight in a water
oven.

Instead of estimating the insoluble acids as just de-
scribed, E. Reichert estimates the volatile fatty-acids and
recommends the following method for the purpose :*

Two and one-half grams of purified water-free fat, pre-
viously filtered through cotton, are placed, in a fluid

*Zeitsch. f. Analyt. Chem. N VIII. 68.

16 QUALITATIVE EXAMINATION OF MILK.

condition, in a flask holding about 150 c. c. (An Erlen-
meyer flask is the best form.) To this add one gram of
solid potassium hydrate and 20 c. c. of 80 per cent alcohol.
This mixture is put on the water bath and agitated fre-
quently, until complete saponification is effected, the
mixture becoming perfectly clear and soapy in appear-
ance. Then, taking the flask from the bath, the soap is
dissolved in 50 c. c. of water-and decomposed by adding
20 c. c. of dilute sulphuric acid (one c. c. of pure sulphuric
acid to 10 c. c. of water). The contents of the flask are
now subjected to distillation. Care must be taken to
prevent the bumping of the fluid, by allowing a light cur-
rent of air to pass through it. It is also recommended to
connect the distilling flask with a large bulbed tube, to
prevent the splashing out of the sulphuric acid. Since the
distillate, especially from poor butter-fats, or by rapid
distillation, always carries with it some solid fatty-acids, it
should be immediately filtered through a moist filter and
collected in a 50 c. c. flask. After 10 to 20 c. c. has dis-
tilled off, pour it back into the flask and again distill,
until the flask contains exactly 50 c. c. of distillate. If the
distillation has been carried on cautiously, the distillate
will be perfectly clear. This distillate is finally trans-
ferred to a larger flask and titrated with tenth-normal
sodium hydrate, using litmus tincture as an indicator.
The titration is considered complete when the blue color
of the litmus remains permanent for some time. Ac-
cording to Reichart, 214 grams of butter-fat require 12.15
c. c. to 14 ¢. c. of tenth-normal sodium hydrate solution.
Figures outside of these limits indicate impure butter-fat.

According to investigations of Pizzi, the milk fat ot
different mammals shows a slight difference with refer-
ence to the amount of volatile fatty-acid contained, which
gives a means of determining the source of the milk,

ALBUMINOIDS. 17

This fact has little practical interest, since in most cases
the analyst has to deal only with cows’ milk.

II. ALBUMINOIDS.

For the examination of milk for the characteristic al-
buminoids, dilute the sample with an equal volume of
water and add hydrochloric acid to a strong acid reaction
and boil. In this way a curdy precipitate of casein and fat
is quickly obtained. This is filtered off and washed on the
filter with water until all the acid is eliminated. Then
to remove the fats wash thoroughly with alcohol and
finally with ether. Subject this material to the follow-
ing reactions which characterize casein.

1. It is readily soluble in very dilute alkali and by
careful addition of acid it is reprecipitated.

2. By treatment with acids in excess, solution takes
place. Itis precipitated from the acid solution by rennet.

3. By boiling the acid or alkali solutions there will
be no precipitation.

4. Oxalates of the alkali metals dissolve it pro-
ducing a milky opalescent fluid. By adding solid mag-
nesium sulphate to the solution a precipitate is formed;
simple dilution of the solution does not effect this pre-
cipitation.

5. Sodium fluoride dissolves casein, especially when
heated. By the introduction of carbon dioxide precipita-
tion takes place.

6. If a sample be placed in a porcelain crucible and
fused with a mixture of sodium carbonate and potassium
nitrate and then cooled and dissolved in dilute nitric acid,
the characteristic reactions of phosphoric acid may be
obtained. (Magnesium mixture, ammonium molybdate.)
(See page 30).

The positive response to the above described reactions
shows conclusively the presence of casein.

18 QUALITATIVE EXAMINATION OF MILK.

III. MILK SUGAR.

To identify the characteristic carbohydrate of milk—
milk sugar or lactose—it first must be isolated in a pure
state. 100 c.c. to 200 c. c. of milk are treated with acetic
acid in the same manner as described, or with rennet to
precipitate the casein, and filtered through a linen cloth.
The filtrate is then heated for ten minutes to coagulate
completely the remaining albuminoids and is again fil-
tered and evaporated to crystallization. After a few days
colorless glistening crystals (rhombic prisms) will have
separated out. The product may be purified by decant-
ing the mother liquor, dissolving in warm water and re-
crystallizing. The following reactions are characteristic
of milk sugar:

1. It reduces an alkaline copper solution (and Fehl-
ing’s solution), red cuprous oxide, CuO, being precipi-
tated. Its reducing power is, however, different from
that of grape sugar (see page 52). To apply this test, one
adds a solution of sodium hydrate to an aqueous solution
of the sugar and then adds drop by drop a solution of
copper sulphate as long as the resulting blue precipitate
dissolves by stirring. By heating this deep blue solution,
red cuprous oxide is precipitated.

2. Lactose also reduces alkaline solutions of bis-
muth, silver and mercury.

3. By boiling a solution of milk sugar for about five
minutes with a slight excess of lead subacetate and add-
ding ammonia to the boiling solution until a permanent
precipitate is obtained, a cherry red solution results.
After a short time a similar colored precipitate settles
out (Rubner).

4. Ifa solution of milk sugar be heated for about
an hour upon the water bath with equal parts of phenyl-
hydrazine and acetic acid (50 per cent.), lactosazone is

MILK SUGAR. 19

formed which on cooling separates out in golden yellow
needles. The same reaction is effected by heating the
sugar solution with two parts of phenylhydrazine hydro-
chloride and three parts of sodium acetate crystals. By
recrystallization out of hot water pure lactosazone may
be obtained, melting at 200 degrees C. (The osazone of
grape sugar, glucosazone, melts at 205 degrees C.)

5. Milk sugar does not undergo alcoholic ferment-
ation directly, but by boiling with dilute sulphuric acid
for an hour and neutralizing with calcium carbonate, the
resulting solution will ferment upon the addition of yeast.

6. Lactose turns the plane of polarized light to the
right, (a) D equals 52.5 degrees. The rotation will be
greater when the solution is boiled for half an hour with
dilute acid and made up to its former volume.

7. Unlike grape sugar, solutions of milk sugar will
not produce cuprous oxide by boiling with copper ace-
tate and acetic acid.

8. The reaction of nitric acid: To 5 grams of the sugar
add 20 c. c. of concentrated nitric acid and cautiously heat
the mixture. A very vigorous reaction takes place, pro-
ducing dense red fumes. When the action of the acid has
ceased the reaction product is allowed to stand in a cool
place for some time. From the solution mucic acid sepa-
rates as acrystallized mass. The crystals are thoroughly
dried, dissolved in ammonia, and the solution then placed
on a water bath and evaporated to dryness. The dried
substance when ‘heated in a test tube yields pyrrol, the
vapor of which will color dark red a pine splinter previ-
ously moistened with hydrochloric acid. Grape sugar,
when treated in a similar manner, with nitric acid yields
oxalic acid but no mucic acid,

Besides the already mentioned difference in the per-
centage of volatile fatty acids contained in the fat of milk
from different animals, one may decide (according to

20 QUALITATIVE EXAMINATION OF MILK.

Pfeiffer) as to the kind of milk, by noting the action
of it when heated; and by the appearance of the curd
formed. For instance, colostrum coagulates upon boil-
ing in large irregular patches. Old cow milk
and old goat milk form firm flakes which quickly become
agglomerated, asses’ milk and mares’ milk yield a flocu-
lent curd, the small soft flakes floating in a turbid milky
fluid. Fresh cow milk, goat milk, sheep milk, asses’
milk, mares’ milk and human milk, a few weeks after de-
livery, will not coagulate by boiling; the latter not even
by addition of dilute (2 per cent.) hydrochloric acid. The
rest coagulate by addition of hydrochloric acid: and
asses’ milk and mares’ milk, as mentioned above, form
small, tender flakes, while cows’, goats’ and sheep’s milk
yield firm flakes suspended in an almost clear fluid.

METHODS OF QUANTITATIVE ANALYSIS.

I. DETERMINATION OF SPECIFIC GRAVITY.

For an absolutely accurate determination of specific
gravity, the pyknometer must be used. The pyknometer
is graduated for a definite temperature. The determina-
tion of specific gravity therefore must be made at that
degree. The milk and the instrument are brought to
the proper temperature by placing in warm water. Since
the cream readily separates from the milk, it is best to
mix the sample thoroughly by shaking before each de-
termination.

The pyknometer is accurately tared, filled with milk and
weighed. Ifthe capacity of the instrument be 10 cc. and
if it weigh, when filled with milk, after deducting the tare,
0.1035 gram, the specific gravity of the milk would be
10 X 0.1035 = 1.035.*

For a less accurate determination of the specific grav-
ity one may use a good areometer (lactodensimeter).
On account of the rapid rise of the cream on milk, one
must read the scale as quickly as possible. An areometer
constructed especially for the examination of milk should
have a scale that will indicate the specific gravity accu-
rately to the fourth decimal place. The range of the

*The pyknometers most commonly used at present, have a
capacity of about 50 c.c.. They are first dried and weighed;
then filled with distilled water at a chosen temperature (usually
17.5° C.) and weighed; and finally rinsed and filled with milk at
exactly the same temperature, and again weighed. :

Weight of milk divided by weight of water equals specific
gravity.—Translators.

21

22 METHODS OF QUANTITATIVE ANALYSIS.

scale should be from 1.025 to 1.040, with the marks indi-
cating the second decimal points at least 20 to 25 m. m.
apart.
In order to obtain the normal specific gravity reading
and thus satisfy the market inspector, skimmed milk is
not infrequently adulterated with water.
~~ In certain cases one may detect this adul-
teration in the following manner: The
milk in question is placed in a narrow, tall
cylinder and shaken thoroughly and tested
with the areometer, and then allowed to
stand quietly for 24 hours for the cream to
rise. After carefully skimming off the
cream, the milk is tested again. For good
cow’s milk (whole milk), the first reading
should be from 1.028 to 1.033 and the sec-
ond from 1.035 to 1.0365. For this purpose
the cream is allowed to separate from the
milk in a vessel provided with a faucet at
the bottom, through which one may readily
draw off the cream-free milk into another
vessel and then test it with the areometer.
The exact composition, however, may be
determined only by a complete chemical
analysis.
The “lactodensimeter” designed by
Quevenne is an areometer with a scale
Fic. 1 of 14 degrees to 42 degrees. The de-

pprer partot@ grees of the scale indicate the specific

2 icpectors, gravity, e. g., 14 represents the specific

gravity 1.014. Hence when the
temperature is 15 degrees C., the reading shows directly
the density of the sample. For temperatures other than
15 degrees C. the proper correction must be made (see

tables pages 23 and 24).

i

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TABLE OF CORRECTIONS FOR THE QUEVENNE

DETERMINATION OF SPECIFIC GRAVITY.

LACTODENSIMETER.

(WHOLE MILK.)

23

8 9 10 | 11 12 | 13 | 14] 15 | 16 | 17 | 18 | 19 | 20
14 | 13.2 | 13.3 | 13.4 | 13.5 | 13.6 | 13.7 | 13.8 | 14.0 | 14.1} 14.2 | 14.4 14.6 | 14.8
15 | 14.2 | 14.3 | 14.4 | 14.5 | 14.6 | 14.7 | 14.8 | 15.0} 15.1) 15.2) 15.4 15.6 | 15.8
16 | 15.2 | 15.3 | 15.4 | 15.5 | 15.6 | 15.7 | 15.8 | 16.0 | 16.1 | 16.3 | 16.5 | 16.7 | 16.9
17 | 16.2 | 16.3 | 16.4 | 16.5 | 16.6 | 16.7 | 16.8 | 17.0 | 17.1] 173 | 17.5 | 17.7 }.17.9
18 | 17.2] 17.3 | 17.4] 17.5] 17.6 | 17.7 | 17.8] 18.0 | 18.1 | 18.3 | 18.5 | 18.7 | 18.9
19 | 18.2 | 18.3 | 18.4 | 18.5 | 18.6 | 18.7 | 18.8 | 19.0 | 19.1 | 19.3 | 19.5 | 19.7 | 19.9
20 | 19.1 | 19.2 | 19.3 | 19.4 | 19.5 | 19.6 | 19.8 | 20.0 | 20.1 | 20.3 | 20.5 | 20.7 ; 20.9
21 | 20.1 | 20.2 | 20.3 | 20.4 | 20.5 | 20.6 | 20.8 | 21.0 | 21.2 | 21.4] 216 , 21.8. 22.0
22 | 21.1 | 21.2 | 21.3 | 21.4 | 21.5 | 21.6 | 21.8 | 22.0 | 22.2 | 22.4 | 22.6 122.8 23.0
23 | 22.1 | 22.2 | 22.3 | 22.4 | 22.5 | 22.6 | 22.8 | 23.0 | 23.2 | 23.4 | 23.6 | 23.8 | 24.0
24 | 23.1 | 23.2 | 23.3 | 23.4 | 23.5 | 23.6 | 23.8 | 24.0 | 24.2 | 24.4 | 24.6 | 24.8 | 25.0
25 | 24.0 | 24.1 | 24.2 | 24.3 | 24.5 | 24.6 | 24.8 | 25.0 | 25.2 | 25.4 | 25.6 . 25.8 | 26.0
26 | 25.0 | 25.1 | 25.2 | 25.3 | 25.5 | 25.6 | 25.8 | 26.0 | 26.2 | 20.4 | 26.6 | 26.9 | 27.1
27 | 26.0 ! 26.1 | 26.2 | 26.3 | 26.6 ; 26.6 | 26.8 | 27.0 | 27.2 | 27.4 | 27.6 | 27.9 ; 28.2
28 | 26.9 | 27.0 | 27.1] 27.2 | 27.4 | 27.6 | 27.8 | 28.0 | 28.2 | 28.4 | 28.6 | 28.9 | 29.2
29 | 27.8 | 27.9 | 28.1 | 28.2 | 28.4 | 28.6 | 28.8 | 29.0 | 29.2 | 29.4 | 29.6 | 29.9 | 30.2
30 | 28.7 | 28.8 | 29.0 | 29.2 | 29.4 | 29.6 | 29.8 | 30.0 | 30.2 | 30.4 | 30.6 | 30.9 | 31.2
31 | 29.7 | 29.8 | 30.0 | 30.2 | 30.4 | 30.6 | 30.8 | 31.0 | 31.2 | 31.4 | 31.7 | 32.0 | 32.3
32 | 30.6 | 30.8 | 31.0 | 31.2 | 31.4 | 31.6 | 31.8 | 32.0 | 32.2 | 32.4 | 32.7 | 33.0 | 33.3
33 | 31.6 | 31.8 | 32.0 | 32.2 | 32.4 | 32.6 | 32.8 | 33.0 | 33.2 | 33.4) 33.7 | 34.0 | 34.3
34 | 32.5 | 32.7 | 32.9 | 33.1 | 33.3 | 33.5 | 33.8 | 34.0 | 34.2 | 34.4 | 34.7 | 35.0 | 35.3
35 | 33.4 | 33.6 | 33.8 | 34.0 | 34.2 | 34.4 | 34.7 | 35.0 | 35.2 | 35.4 35.7 | 36.0 | 36.3

24

TABLE OF CORRECTIONS FOR THE QUEVENNE

METHODS OF QUANTITATIVE ANALYSIS.

LACTODENSIMETER.
(SKIMMED MILK.)

17.3
18.3
19.3
20,3
21.3
22.3
23.2
24.1
25.1
26.1
27.1
28.1
29.1
30.1
31.1
32.1
33.1
34.0
35.0
36.0
37.0
37.9
38.8

17.4
18.4
19.4
20.4
21.4
22.4
23.3
24.2
25.2
26.2
27.2
28.2
29.2
30.2
31.2
32.2
33.2
34.1
35.1
36.1
37.1
38.0
38.9

17.5
18.5
19.5
20.5
21.5
22.5
23.4
24.2
25.3
26.3
27.3
28.3
29.3
30.3
31.3
32.3
33.3
34.2
35.2
36.2
37,2
38.2
39.1

17.6
18.6
19.6
20.6
21.6
22.6
23.5
24.4
25.4
26.4
27.4
28.4
29.4
30.4
31.4
32.4
33.4
34.3
35.3
36.3
37.3
38.3
39.2

17.7
18.7
19.7
20.7
21.7
22.7
23.6
24.5
25.5
26.5
27.5
28.5
29.5
30.5
31.5
32.5
33.5
34.4
35.4
36.4
37.4
38.4
39.4

17.8
18.8
19.8
20.8
21.8
22.8
23.7
24.6
25.6
26.6
27.6
28.6
29.6
30.6
31.6
32.6
33.6
34.6
35.6
36.6
37.6
38.6
39.6

17.9

18.9"

19.9
20.9
21.9
22.9
23.9
24.8
25.8
26.8
27.8
28.8
29.8
30.8
31.8
32.8
33.8
34.8
35.8
36.8
37.8
38.8
39.8

18.1
19.1
20.1
21.1
22.1
23.1
24.1
25.1
26.1
27.1
28.1
29.1
30.1
31.2
32.2
33.2
34.2
35.2
36.2
37.2
38.2
39.2
40.2

18.2
19.2
20.2
21.2
22.2
23.2
24.2
25.2
26.3
27.3
28.3
29.3
30.3
31.4
32.4
33.4
34.4
35.4
36.4
37.4
38.4
39.4
40.4

18.4
19.4
20.4
21.4
22.4
23.4
24.4
25.4
26.5
27.5
28.5
29.5
30.5
31.5
32.6
33.6
34.6
35.6
36.6
37.6
38.6
39.6
40.6

18.6
19.6
20.6
21.6
22.6
23.6
24.6
25.6
26.7
27.7
28.7
29.7
30.7
31.8
32.8
33.8
34.8
35.8
36.9
37.9
38.9
39.9
40.9

18.8
19.8
20.8
21.8
22.8
23.8
24.8
25.8
26.9
27.9
28.9
29.9
30.9
32.0
33.0
34.0
35.0
36.0
37.1
38.2
39.2
40.2
41.2

DETERMINATION OF ACIDITY. 25

In this table the figures in the top horizontal line indi-
cates the temperature of-the milk and those in the left
vertical line indicate the lactodensimeter reading. For
instance, if the areometer reading is 30 and the tempera-
ture is 16 degrees, the exact specific gravity may be found
in the ninth column and the sixth line from the bottom.
This figure 30.2 signifies a specific gravity of 1.0302.

Other forms of apparatus described by Bischoff, Reck-
nagel and Soxhlet, do not require the table of correc-
tions. They are provided with a thermometer without
the regular thermometric scale. The thermometer read-
ing is simply a correction number which must be either
added to or subtracted from the reading on the main
scale (see figure 1).

II. THE DETERMINATION OF THE REACTION AND
ACIDITY.

The reaction of human milk in its normal state is alka-
line; the milk of carniverous animals is acid. Cow’s milk
should, according to earlier investigators, has an am-
photeric reaction. However, in a perfectly fresh condi-
tion, that is, at the moment it leaves the udder, cow’s milk
reacts slightly acid (Vaudin). The reaction does not
change if the milk is placed in a hermetically sealed glass
tube and heated a long period at a temperature of 100
‘degrees C. It becomes more strongly acid when allowed
to stand in the open air or even with the air excluded.
Through the action of bacteria (ferments) milk sugar is
converted into lactic acid (commonly called lactic acid
fermentation). Ordinarily inactive lactic acid is thus
produced, although the formation of active paralactic
acid has been observed by Gunther and Thierfelder. Oc-
casionally carbonic acid and ethyl alcohol are formed
(Leichmann). The acid production, which is favored by
high temperatures, ceases when a fixed amount of lactic

26 METHODS OF QUANTITATIVE ANALYSIS.

acid has been formed (0.604 per cent., according to
Timpe), because the ferment is destroyed when the de-
composition reaches a certain degree of concentration
wherein the conditions for its growth are removed.

In order to ascertain the age of milk from the strength
of the acid reaction, the initial reaction must be known.
If the souring has advanced so far that it may be tasted
the casein will soon begin to coagulate. The milk is then
doubtless old and spoiled. Before this stage is reached,
however, one may determine the age of milk with some
degree of accuracy, in the following manner: old milk

.coagulates by treating with carbon dioxide and sub-
sequently heating to boiling. Older milk, without treat-

-ment with carbon dioxide coagulates by boiling, while
with still older milk coagulation takes place without
boiling by simply forcing in carbon dioxide. Quite fresh
milk, especially cow milk at the beginning of her lactation
period, will sometimes curdle by boiling, but the result-
ing curd in this case consists not of casein, but of another
proteid, viz., globulin. If this point is to be proved, the
sample in question is treated with di-sodium phosphate
solution till the acidity is almost neutralized, and then
boiled. Under these conditions albumin and globulin
alone will be precipitated out, if the milk is fresh. On the
other hand, if the sample should curdle by boiling with-
out adding di-sodium phosphate it is evident that the
milk is old and that fermentation has begun, forming lac-
tic acid. The curd in this case is casein. When alkaline
or neutral milk is boiled the curd produced is always
albumin.*

*Sembritzki found that by continually removing the scum
that forms on boiling milk, he could obtain 1.023 per cent of the
milk in this way. Since albumin is not present in milk in such
large quantity, it follows that some other body—probably case-
in—must enter into the composition of this scum or curd. See

“Poggendorf’s Annalen der Physik und Chemie,” 37, page 460.)
Translators.

DETERMINATION OF ACIDITY. 27

For the quantitative estimation of acidity of milk, a
measured quantity of the sample is titrated with dilute
sodium hydrate of known strength; 10 c. c. of milk is di-
luted with go c.c.of distilled waterand thoroughly shaken.
Add one or two drops of a strong alcoholic solution of
phenolphthalein for an indicator. From a burette (gradu-
ated in one-tenth c. c.), a one-tenth or one-fifth normal
solution of sodium hydrate is run in drop by drop with
constant stirring, until the bright red color remains per-
manent. (Phenolphthalein is colored red with alkalies.)
The operation is repeated with a second portion of the
milk and the mean of the two titrations taken. The re-
sults should not differ by more than one-tenth c. c. The
acidity is expressed in terms of cubic centimeters of so-
dium hydrate required. (One should not accept the re-
sults of one titration.) According to Pfeiffer, the quantity
of acid in different kinds of milk differs according to the
time of year, the climate, and other conditions, so that no
limits can be fixed. He therefore recommends that every
observer find the average acidity coefficient for milk from
his locality, by making numerous tests of strictly pure
samples, “preferably ‘stall samples.’ ”

The age of milk may be approximately judged by de-
termining the acidity in the following manner: The
method is not considered very reliable. 25 c. c. of milk are
placed in the flask and a few drops of phenolphthalein
added and titrated, as above, with barium hydroxide solu-
tion until a faint red tint remains; 1 c.c. of baryta solu-
tion should be equivalent to 0.005 grams SO, that is,
one liter of the solution must contain 10.705 grams of
barium hydrate. With fresh milk the red color appears on
adding 17 c.c. of the barium hydrate solution. This acid-
ity remains constant at 10 degrees Centigrade for 48
hours, at 15 degrees for 20 hours and at 37 degrees for 5
hours. If milk kept for one hour in a culture oven

28 METHODS OF QUANTITATIVE ANALYSIS.

shows after that time an increased acidity, it was not
fresh and should not be used as food for children.

One may confirm his opinion as to the age of the milk
in question by the use of a eudiometer tube. The tube is
filled with milk, and the open end placed under mercury
to exclude the air. If the milk be old, bubbles of gas
will collect in the upper end of the tube. Pure fresh
milk after standing 12 hours will develop no perceptible
amount of gas. (Schaffer.)

III. DETERMINATION OF TOTAL SOLIDS.

A platinum crucible with the cover (or in the absence
of this, a porcelain dish with cover) is accurately weighed
and into it is measured five or ten c.c. of the sample which
has been thoroughly shaken to render it homogenous.
The dish and contents are then placed in a water oven and
dried at 100 degrees C. The cover is placed on the dish
in such a manner as to allow the escape of steam without
permitting dust to enter. After 15 to 20 hours the dish
is removed from the oven and allowed to cool.over sul-
phuric acid in a desiccator, and weighed. It is then
placed in the drying oven again for half an hour and once
more cooled and weighed. If there be no difference be-
tween the first and second weighing, the determination
may be considered completed. Otherwise the heating
must be continued until the weight becomes constant.
By deducting the weight of the empty dish from this
last weight obtained, the weight of the residue or dry
matter is determined. If it be desired to weigh the milk
instead of measuring it, the sample is placed in a tared
crucible, and weighed quickly. The cover should be
kept in place to avoid evaporation. The desiccation of
milk by mixing with some porous substance such as gyp-
sum or pumice, or with sand, or by absorbing it with
paper, is not to be recommended. Such treatment inter-

DETERMINATION OF ASH. 29

feres with the accuracy of the ash determination which
usually follows the estimation of total solids.

By prolonged heating the albuminoids of milk acquire
a brown color (Renck), and according to Cazeneuve and
Haddon and Renck the lactose becomes caramelized, so
that the dry residue is always yellowish brown in color.
It has been shown that this is not a source of error.

The dry matter is very hygroscopic and therefore the
dish must be tightly covered while being weighed.

IV. DETERMINATION OF ASH.

To determine the ash or mineral substances of milk
one may use to advantage the residue of dry matter just
obtained. This residue is heated to dull redness in the
dish loosely covered. After igniting for one to two
hours the ash becomes pure white. The crucible and
contents are then cooled in the desiccator and weighed.
If phosphoric acid in the ash is to be determined, not
less than 20 c.c. of milk should be incinerated. The ash
is dissolved in very dilute nitric acid and treated with an
excess of ammonium molybdate solution. (At least 4o
parts of molybdate to one part of phosphoric acid). A
yellow precipitate of ammonium phosphomolybdate is
obtained. The whole is then allowed to stand for twelve
hours at a temperature of about 40 degrees C., and is
then filtered through a small filter and the filtrate exam-
ined. An addition of ammonium molybdate to the fil-
trate should cause no further precipitation, even on long
standing. The precipitate on the filter is washed with
a 10 per cent. solution of ammonium nitrate until the
washings cease to react acid and is then dissolved by add-
ing a little warm dilute ammonia. Rinse the filter thor-
oughly with very dilute ammonia. The filtrate and
washings containing the ammonium phosphomolybdate

30 METHODS OF QUANTITATIVE ANALYSIS.

in solution are collected in the beaker and treated with
ammonium chloride (about one-sixth its volume). To
this is added while stirring constantly magnesia mixture
as long as a precipitate is formed. Finally a slight excess
of magnesia mixture is run in and the solution allowed to
stand for 12 hours in the cold. The precipitate of am-
monium magnesium phosphate is then filtered off and
washed with diluted ammonia (1 to 3) until a sample of
the filtrate acidified with nitric acid gives no reaction with
silver nitrate solution. The filter and contents are then
dried at 100 degrees and as much as possible of the pre-
cipitate is removed from the paper. The filter is inciner-
ated in a weighed porcelain crucible and the precipitate
is then added andthe whole ignited at a bright red heat.
By this ignition magnesium pyrophosphate is formed,
ammonia and water being expelled. If on cooling the
residue appears gray, moisten with a few drops of nitric
acid, then drive off the acid carefully by heating and again
ignite. By this treatment a pure white mass should
remain. One part of magnesium pyrophosphate is
equivalent to 0.6375 part of phosphoric acid (P,O,).

Ammonium molybdate solution is prepared in the fol-
lowing manner: 100 grams of pure molybdic acid are dis-
solved in 400 grams of ammonia (specific gravity 0.960,
equivalent to 10 per cent. NH,). This solution is poured
cautiously into 1500 grams of nitric acid (specific grav-
ity 1.2). The mixture is warmed for about an hour at
a temperature not to exceed 50 degrees C. and then kept
for two or three days in a moderately warm place. In
case molybdic acid precipitates out, the solution should
be filtered.

Preparation of magnesia mixture: 55 grams of mag-
nesium chloride and 105 grams of ammoniam chloride
are dissolved in water, 350 c.c. of armmonia (24 per cent.,
specific gravity 0.91) added and the solution diluted to

DETERMINATION OF FATS. 31

Fic. 2.
The Soxhlet ex-

traction ap-
prratus.

one liter. The solution is kept in a tight-
ly stoppered bottle for a few days and is
then filtered.

V. DETERMINATION OF THE PER
CENT OF FATS.

For the determination of fats a large
number of methods have been pro-
posed, but after a careful examination
of the same there are only a few that can
be recommended. Of these the method
of Liebermann-Weiss is especially com-
mendable for its simplicity of detail. The
Soxhlet aerometric method, while it is
only applicable to cow's milk, is very sat-
isfactory, and may be carried out without
difficulty and with little expense of time.
The apparatus, however, is not very
cheap.

A. SOXHLET’S GRAVIMETRIC METH-
OD OF ANALYSIS. (Applicable to
all kinds of milk.)

Ten c. c. of the sample are placed ina
small porcelain dish containing enough
pure sea sand to absorb the milk. The
dish and sand should have been pre-
viously heated to redness. The dish is
placed on the water bath, and the con-
tents stirred and evaporated until the
sand appears to be dry. This will be ac-
complished inabout half anhour. Theob-
ject in keeping the contents well stirred
is to prevent the agglomerated sand from
adhering to the walls of the dish. The
contents of the dish are carefully trans-

32 METHODS OF QUANTITATIVE ANALYSIS.

ferred, aided by a folded piece of paper, to the extractor.
The dish as well as the glass rod and the paper used are
finally rinsed with ether into the extraction apparatus.
The Soxhlet extraction apparatus is composed of the fol-
lowing parts: see figure 2. The large tube (a) is
sealed across at the bottom and is intended to hold the
material to be extracted. Into the lower end a narrow
tube (b) is sealed and bent upward parallel to tube (a).
At the point opposite about the middle of (a), this small
tube is curved downward and the end sealed into the
stem (d) of the extractor. The tube (e) connects the
stem (d) with the upper part of the extractor (a). In the
lower end of a tube (a) is placed in a tuft of fat-free
cotton (about 2 cm. high). Upon this the substance to be
analyzed is placed and the apparatus is connected by
means of a tightly fitting cork with a wide necked flask
of about 150 c.c. capacity in which is contained about 75
or 80 c. c. of ether. The upper end of the extractor is con-
nected with a Liebig condenser (g). The flask is now
warmed on the water bath. The ether vapor passes
through the tubes (d) and (e) into the condenser (g). The
condensed ether drops back into the tube (a) containing
the substance to be extracted. Because of the connec-
tion of the tube (b) with (a), the ether is always equally
high in both tubes. When the ether reaches the highest
point at (h) the tube (b) acts as a siphon and draws the
ether, containing the fat in solution, into the flask below.
The ether again vaporizes and fills the extractor and
siphons off. Thus the process may be continued in-
definitely.

The temperature of the water bath is so regulated that
within one hour the tube (a) is filled and emptied about
twelve times. At the end of this time the extraction may
be considered completed. The temperature of the water
bath should not be too high, because,.if the ether boils

DETERMINATION OF FATS. 33

too rapidly the vapor will not be entirely condensed, and
a considerable part of it will thus be lost. The quantity of
ether used must be sufficient to fill the tube (a) to the
top of the siphon tube (b) at least one and a half times.
The different parts of the apparatus should be connected
by tightly fitting corks or by ground glass joints.

The flask containing the fat in ether solution is placed
on the water bath and the ether distilled off. It is then
placed in drying oven and dried at 105 degrees C. to con-
stant weight. The weight of the empty flask having been
previously ascertained, the increase in weight will indi-
cate the weight of fat in the sample. From this data the
per cent of fats may be readily calculated.

Instead of evaporating with sand on the water bath—
a process which takes more or less time—lFernandez-
Krug and Hampe recommend the mixing of five or ten
c.c. of milk with 7.5 to 15 grams of powdered kaolin and
5 to 10 grams of water-fee sodium sulphate. In this way
a perfectly dry mass is obtained which may be extracted
at once with ether.

Pfeiffer suggests precipitating out the albuminoids and
fat of the milk and filtering through a folded filter. The
residue is washed a few times with water, dried at 100
degrees and placed with the filter in a Soxhlet extraction
apparatus. To precipitate the fats and albuminoids
trichloracetic acid or copper hydrate may be used.

B. THE EXTRACTION OF FATS BY MEANS OF PE-
TROLEUM ETHER. (The Lieberman-Weiss Method.)

Liebermann and Szekely and Weiss propose petro-
leum ether as a solvent in determining the per cent of
fat. The method is applicable to cows’ and human milk
and may be carried out rapidly without the use of a
great deal of apparatus. A glass cylinder about 15 cm.

34 METHODS OF QUANTITATIVE ANALYSIS.

high and about 4 cm. in diameter is chosen and pro-
vided with a tightly fitting cork or a glass stopper; 50
c. c. of the milk are placed in the cylinder and treated
with 5 c. c. of a potassium or sodium hydrate solution
and mixed by shaking. To this is added 50 c. c. of
petroleum ether of low boiling point, and 50 c. c. of alco-
hol (96 per cent). This mixture is shaken two or three
times for three minutes and then allowed to stand for
half an hour. The upper one of the three noticeable
layers consists of petroleum éther with the fat in solu-
tion, With the aid of a graduated pipette an aliquot
proportion of this solution is drawn off and transferred
to a small weighed flask. The petroleum ether is evap-
orated and the residue of fat is dried at 100 degrees and
weighed. By a simple calculation the percentage of fat
in the sample is easily determined. For instance, if 25
c. c. of the petroleum ether solution be drawn off and
evaporated and one gram of fat be thus obtained, 100
parts of milk will then contain I x 2x 2 = 4 parts of fat,
or 4 per cent.

This method has the advantage that the analyst may
carry on several control tests with the same original milk
sample. It is especially valuable where a very large
number of fat estimations are to be made. The results
accord very well with the Soxhlet areometric and gravi-
metric methods. A simple process, a modification of the
above, is described by Hoppe and Seyler. It is said to
yield good results. Instead of petroleum ether, the
authors employ ordinary ethyl ether. Liebermann has
criticized this method on the ground that ethyl ether not
only dissolves fats but other constituents of milk. A
series of experiments made by the author shows that
there is scarcely an appreciable difference in action be-
tween the two reagents. It may be said in favor of the
cheaper fluid, petroleum ether, that it seems to be more

U

DETERMINATION OF FATS. 25

readily removed than the ethyl ether. In any case the
gravimetric methods just described are by far the sim-
plest and most quickly carried out.

C. SOXHLET’S AEROMETRIC METHOD. (Applicable
only to cow’s milk.)

The method depends upon the principle of estimating
the specific gravity of the ether solution of the fats. A
measured quantity of milk is treated with potassium
hydrate solution and ether. By shaking, the fat is taken
up by the ether, and the specific gravity of the resulting
solution determined in a special apparatus. Tor this
process the following reagents and equipment are neces-
sary:

1. Potassium hydrate solution, specific gravity 1.27
(400 grams of solid caustic potash are dissolved in water
and after cooling, the solution is made up to one liter).

2. Ethyl ether, purified by shaking with one-tenth to
two-tenths volume of water, and pouring off the clear
liquid.

3. Ether, commercial ethyl ether. (Need not be puri-
fied.)

4. <A vessel of about four liters capacity to be filled
with water at 17 to 18 degrees C.

5. Three pipettes; one 200 c. c., one 60 c. c., and one
IO c. ¢. capacity.

6. Several milk flasks of one-half liter capacity.

7. Two accurate areometers with thermometers: one
for skimmed milk, and one for whole milk. These spin-
dles are to be used in a specially constructed cylinder
which is kept surrounded with cold water.

8. A rubber hand bellows.

Details of the Method: The milk is
brought to a temperature of 18 degrees by holding the
bottle in water of the proper temperature. The sample

36 METHODS OF QUANTITATIVE ANALYSIS.

is well shaken and 200 c. c. of it transferred to a milk
flask. It is then treated with 10 c. c. of the potassium
hydrate solution and mixed by shaking. To this 60
c. c. of ether, previously saturated with water, are added
and the flask then quickly stoppered. The flask is al-
lowed to stand in-a water bath at 18 degrees for 15 min-
nutes, and during this time it is shaken every half minute
(three or four vertical tosses each time). In this amount
of time the ether solution of the fats usually separates out
clear, but in case of milk very rich in fat a longer time is
required.

The method of procedure may be best explained by
reference to the illustration (Fig. 3).

The glass cooler (A) is filled with water at 18 degrees
C. The milk flask (B) which contains the ether-fat solu-
tion is provided with a two-hole stopper. Into one of
the holes is fitted the bent glass tube (C), allowing it to
protrude just through the cork. The other end of the
tube is connected with the hand bellows (D). Through
the other hole of the cork is put a longer glass tube
(E) also bent to a right angle. The tube should extend
in the bottle almost to the bottom of the layer of ether
solution. The end of the tube outside is connected
with the stem of the areometer cylinder (F) by a rubber
tube. The pinchcock (H) serves to hold the ether solu-
tion in (F).

The Operation: By removing the stopper (L)
from the cylinder and opening the stopcock (H), the
fat solution is forced into the cylinder (F) by pressure
from the hand bellows. The cylinder is filled until the
areometer (G) floats freely, when the stopcock is quickly
closed. By adjusting the screw in the foot of the sup-
port the apparatus is brought to a vertical position. The
areometer and thermometer readings are then taken and
by referring these to the table following, the per cent of

TABLE SHOWING THE PERCENTAGE BY WEIGHTS OF
FATS IN WHOLE MILK AS DETERMINED FROM
THE OTHER SOLUTION AT 17.5°C.

(SOXHLET’S TABLE.)

Spec. Fat |Spec. Fat |Spec. Fat |Spec. Fat |Spec. Fat
Grav. Per Cent] Grav. Per Ct. |Grav. Per Ct.|Grav. Per Ct./Grav. Per Ct.
43.0 2.07 47.7 2.61 | 52.3 3.16 | 56.9 3.74 | 61.5 4.39
43.1 2.08 47.8 2.62 | 52.4 3.17 | 57.0 3.75 | 61.6 4.40
43.2 2:09 47.9 2.63 | 52.5 3.18 | 57.1 3.76 | 61.7 4.42
43.3 2.10 48.0 2.64 | 52.6 3.20 | 57.2 3.78 | 61.8 4.44
43.4 oll 48.1 2.66 | 52.7 3.21 | 57.3 3.80 | 61.9 4.46
43.5 ald 48.2 2.67 | 52.8 3.22 | 57.4 3.81 | 62.0 4.47
43.6 213 48.3 2.68 | 52.9 3.23 | 57.5 3.82 | 62.1 4.48
43.7 2.14 48.4 2.70 | 53.0 3.25 | 57.6 3.84 | 62.2 4.50
43.8 2.16 48.5 2.71 | 53.1 3.26 | 57.7 3.85 | 62.3 4.52
43.9 2.17 48.6 2.72 | 53.2 3.27 | 57.8 3.87 | 62.4 4.53
44.0 2.18 48.7 2.73 | 53.3 3.28 | 57.9 3.88 | 62.5 4.55
44.1 219 48.8 2.74 | 53.4 3.29 | 58.0 3.90 | 62.6 4.56
44.2 2.20 48.9 2.75 | 53.5 3.30 | 58.1 3.91 | 62.7 4.58
44.3 2.22 49.0 2.76 | 53.6 3.31 | 58.2 3.92 | 62.8 4.59
44.4 2.23 49.1 2.77 | 53.7 3.33 | 58.3 3.93 | 62.9 4.61
44.5 2.24 49.2 2.78 | 53.8 3.34 | 58.4 3.95 | 63.0 4.63
44.6 2:25 49.3. 2.79 | 53.9 3.35 | 58.5 3.96 | 63.1 4.64
44.7 2.26 49.4 2.80 | 54.0 3.37 | 58.6 3.98 | 63.2 4.66
44.8 2:27, 49.5 2.81 | 54.1 3.38 | 58.7 3.99 | 63.3 4.67
44.9 2.28 49.6 2.83 | 54.2 3.39 | 58.8 4.01 | 63.4 4.69
45.0 2.30 49.7 2.84 | 54.3 3.40 | 58.9 4.02 | 63.5 4.70
45.1 2.31 49.8 2.86 | 54.4 3.41 | 59.0 4.03 | 63.6 4.71
45.2 2.32 49.9 2.87 | 54.5 3.43 | 59.1 4.04 | 63.7 4.73
45.3 2.33 50.0 2.88 | 54.6 3.45 | 59.2 4.06 | 63.8 4.75
45.4 2.34 BO.1 2.90 | 54.7 3.46 | 59.3 4.07 | 63.9 4.77
45.5 2.35 50.2 2.91} 54.8 3.47 | 59.4 4.09 | 64.0 4.79
45.6 2.36 50.3 2.92 | 54.9 3.48 | 59.5 4.11 | 64.1 4.80
45.7 2.37 50.4 2.93 | 55.0 3.49 | 59.6 4.12 | 64.2 4.82
45.8 2.38 50.5 2.94 | 55.1 3.51 | 59.7 4.14 | 64.3 4.84
45.9 2.39 50.6 2.96 | 55.2 3.52 | 59.8 4.15 | 64.4 4.85
46.0 2.40 50.7 2.97 | 55.3 3.53 | 59.9 4.16 | 64.5 4.87
46.1 2.42 50.8 2.98 } 55.4 3.55 | 60.0 4.18 | 64.6 4.88
46.2 2.43 50.9 2.99 | 55.5 3.56 | 60.1 4.19 | 64.7 4.90
46.3 2.44 51.0 3.00 | 55.6 3.57 | 60.2 4.20 | 64.8 4.92
46.4 2.45 SLA. 3.01 | 55.7 359 | 60.3 4.21 | 64.9 4.93
46.5 2.46 51.2 3.03 | 55.8 3.60 | 60.4 4.23 | 65.0 4.95
46.6 2.47 51.3 3.04 | 55.9 3.61 | 60.5 4.24 | 65.1 4.97
46.7 2.49 51.4 3.05 | 56.0 3.63 | 60.6 4.26 | 65.2 4.98
46.8 2.50 51.5 3.06 | 56.1 3.64 | 60.7 4.27 | 65.3 5.00
46.9 2.51 51.6 3.08 | 56.2 3,65 | 60.8 4.29 | 65.4 5.02
47.0 2.52 51.7 3.09 | 56.3 3.67 | 60.9 4.30 | 65.5 5.04
47.1 2.54 51.8 3.10 | 56.4 3.68 | 61.0 4.32 | 65.6 5.05
47.2 2.55 51.9 3.11 | 56.5 3.69 | 61.1 4.33 | 65.7 5.07
47.3 2.56 | 52.0 3.12 | 56.6 3,71 | 61.2 4.35 | 65.8 ‘5.09
47.4 2.57 52.1 3.14 | 56.7 3.72 | 61.3 4.36 | 65.9 5.11
47.5 2.58 52.2 3.15 | 56.8 3.73 | 61.4 4.37 | 66.0 5.12

47.6 2.60

DETERMINATION OF FATS. 37

fats is determined. At a temperature of 1714 degrees C.
no correction is necessary. Otherwise, for each one-tenth
degree above this point add to the areometer reading
one degree, or for each one-tenth below, deduct one
degree.

For very poor milk (low in fats), at times, a clear ether

} iN
Fa Ma ae
a4, \ no}
Me
E Ti
(4
i il
al
I {
: |
A
2 | 8
SON EN
OO
Fic. 3.

fat solution will not separate out, but forms a gelatinous
mass. In such cases Soxhlet suggests the addition of
from 20 to 25 drops of a soap solution before mixing
with ether. The soap solution for this purpose 1s
prepared by heating on a water bath 15 grams
of stearic acid! with about 25 c. c. of alcohol (96 per cent)

‘An ordinary stearin candle will answer the purpose.

38 METHODS OF QUANTITATIVE ANALYSIS.

and toc. c. of the above mentioned potassium hydrate so-
lution. Continue heating until a clear yellow solution is
obtained, and then dilute to 100 c. c.

D. METHODS WHICH GIVE ONLY APPROXIMATE-
LY ACCURATE RESULTS.

A large number of methods and apparatus have been
proposed for the rapid estimation of fats, but most of
them are accurate only to a moderate degree. To this
class belong the “cremometer,” the “lactoscope,” the
“lactocrite,” and others. The first of these depends upon
the principle of measuring the layer of cream that sepa-
rates out; the others are optical methods. The cream
measure (cremometer) would give reliable results, if in
each case, equal volumes of cream of the same fat con-
tent would separate out when samples of milk of uniform
fat percentage are allowed to stand in proper vessels un-
der similar conditions. But this is not the case, and
moreover, the temperature, the kind of vessel, and the
condition of the milk, all have an influence upon the
amount of cream obtainable. Thus for example, milk
diluted with water, furnishes proportionately more

--cream than the same milk undiluted, because the fat
globules in the watered milk can rise to the surface
more easily than in the pure milk, which is specifically
heavier.

For similar reasons the optical methods for the more
exact determinations should be rejected. The trans-
parency of milk bears a certain relation to the number
of fat globules contained in it. That is to say, the larger
the number of fat globules, the greater will be the opac-
ity of the milk column. If now the number of fat glo-
bules were proportional to the fat content, this method,
based upon the transparency of milk, could not be crit-
ivized. But this is not the case.

TABLE SHOWING THE PERCENTAGE BY WEIGHT OF
FATS IN SKIMMED MILK, AS DETERMINED FROM
THE SPECIFIC GRAVITY OF THE ETHER
SOLUTION AT 17.35° C.

(SOXHLET’S TABLE.)

Spec. Fat |Spec. Fat |Spec. Fat leinae: Fat (Spec. Fat
Grav. Per Ct.|Grav. Per Ct./Grav. Per Ct.:Gray. Per Ct..\Grav. Per Ct.
21.1 0.00 | 25.5 0.41 | 29.9 0.82 | 34.3 1.22 | 38.7 1.64
21.2 0.01 | 25.6 0.42 | 30.0 0.83 | 34.4 1.23 | 38.8 1.65
21.3 0.02 | 25.7 0.43 | 30.1 0.84 | 34.5 1.24 | 38.9 1.66
21.4 0.03 | 25.8 0.44 | 30.2 0.85 | 34.6 1.24 | 39.0 1.67
21.5 0.04 | 25.9 0.45 | 30.3 0.86 | 34.7 1.25 | 39.1 1.68
21.6 0.05 | 26.0 0.46 | 30.4 0.87 | 34.8 1.26 | 39.2 1.69
21.7 0.06 | 26.1 0.47 | 30.5 0.88 | 34.9 1.27 | 39.3 1.70
21.8 0.07 | 262 0.48 | 30.6 0.88 | 35.0 1.28 | 39.4 eval
21.9 0.08 | 26.3 0.49 | 30.7 0.89 | 35.1 1.29 | 39.5 ive
22.0 0.09 | 26.4 0.50 | 30.8 0.90 | 35.2 1.30 | 30.6 173
22.1 0.10 | 26.5 0.50 | 30.9 W.91 . 35.3 1.31 | 39.7 17
TS 0.11 | 26.6 0.51 | 31.0 0.92. 35.4 1.32.) 30.8 1.7:
22.3 0.12 | 26.7 0.52 | 31.1 0.93 | 35.5 1.33 | 39.9 is
22 4 0.13. | 26.8 0.53 | 31.2 0.94 35.6 133) 40.0 1
22.5 0.14 | 26.9 0.54 | 31.3 O95 38.7 1.34 | 40.1 1.78
93.6 ~ 0.15 | 27.0 0.55 | 31.4 0.95 35.8 1.35 | 40.2 1.79
22.7 016 | 27.1 0.56 | 31.5 U.96 , 35.9 1.45 1 40,3 1.80
22.8 0.17 | 27.2 0.57 | 31.6 0.07 | 36.0 1.3% | 40.4 1st
224 0.18 273 0.58 | 31.7 0.98 . 36.1 1.38 40.5 LA
23.0 0.19 | 27.4 0.59 | 31.8 0.09 30.2 1.39 40.6 1.83
23.1 0.20 | 27.5 0.60 | 31.9 1.00 | 30.3 1.40 | 40.7 1.84
23.2 0.21 | 27.6 0.60 | 32.0 1.01 | 36.4 1.41. 40.8 1.85
23.3 Hae ee: | wet. | aa 102! 3058 142] 40.9 1.86
23.4 0.23 | 27.8 0.62 | 32.2 1.03 | 30.6 1.48 | 41.0 Tesi
23.5 0.24 | 27.9 0.63 | 32.3 1.04 | 30.7 144) 411 1.88
23.6 U.as: } 2a.0 0.04 | 32.4 1.08 | 30.8 1.45 1 41.2 1.49
23.7 0.25 | 28.1 0.63 | 32.5 1.08 | 30.9 La 44.3 1.90
23.8 i. | B88 d.00 | 32.6 1.06 | 37.0 eee 1.91
23.9 u.o7 | O38 G67 | 327 op! 8e1 1.48 ahs 1.92
24.0 O28 | 28.4 0.68 | 32.8 1.08 | 37.2 1.49 41.6 1.93
24.1 0.29 | 28.5 0,09 | 32.9 1.09 | 373 1.50 41.7 1.94
24.5 v.30 | 28.6 0.70 | 33.0 1.10 | 37.4 1.51 | 41.8 1.95
24.3 0.30 | 28.7 0.71 | 33.1 1At |. 3n8 152° 41.9 1.96
ad 0-31) 28.8 0.52» 33.3 {sy Sas 1.53 42.0 1.97
24.5 0.32 | 28.9 OFF 33S 113) Shar 13h. 2a 1.08
24.6 0.33 | 20,0 Oc | 33.4 1d | ans 1.55 42.2 1.99
24.7 0.34 | 29.1 0.75. 33.5 1.15 , 37.9 1.56 42.3 2.00
24.8 0.35 | 292 0.76 | 33.6 1.15 38.0 1S; A 2.01
24.0 0.30 | 203 O47 83.3 1.16 | 38.1 1.58 42.5 2.02
25.0 0.37. 29.4 O78 ak 1.17, 382 1.59 42.6 2.03
25.1 W.38 29.5 0.79 33.9 1.18 | 33.3 Lod 42.7 2.04
98, 2 O89 | 200 OS. 34.0 1.19 3N.4 1.61 42.8 2.05
253 odo aoe ee ee Lay 28.3 Lat 42.9 2.06
23.4 O40 20.8 wal 342 1.21 | 88.6 Lint 430 20

|
I
i
'

DETERMINATION OF FATS. 39

The optical methods have, moreover, the disadvantage
that they are dependent upon the individual perception
of each observer. Naturally each eye is not equally sen-
sitive in detecting differences of luminosity. Outside
conditions under which the test is carried on, also exert
an influence on the result. Even for one and the same
eye the sensitiveness of the test is
not the same in bright sunlight as
in cloudy weather, nor in artificial
light, as in daylight, etc. Although
these methods are not practicable
for very accurate work, they are
useful to confirm the doubtful re-

HY

= 0 sults obtained by more exact deter-
= minations. They are valuable to
=i the physician, the pharmacist, and
[S40 especially to the market inspectors,
= % to detect occasional adulteration.

The processes have the further ad-
vantage that the observer need not
| be especially skilled. For a close
decision of the quality of a sample,
however, they are not suited, for
the reason just stated.

The cremometer of Chevalier

Ties: (Fig. 4) consists of a glass cylinder
The Chevalier ‘ “i "
Gremometer: 20 cm. high and 4 cm. in diameter,

with a capacity of 160 ¢c. ¢.

It is provided with a hundred-point scale, the zero
mark of which is placed 5 cm. below the top rim. The
graduations usually are made only from zero degrees to
50 degrees. For the test, the apparatus is filled to the
zero mark with milk and allowed to stand for four hours
in a room where the temperature is moderate and uni-

40 METHODS OF QUANTITATIVE ANALYSIS.

form. The cream layer is then measured on the scale.
For whole milk the average is 10 to 15 degrees and for

Fie. 5.

The Feser
Lactoscope.

half-skimmed milk, 5 to 8 degrees. In
order to decide from the reading whether
the milk has been skimmed or watered,
one must determine the density of the
sample thus freed from cream by the
method given in the chapter on specific
gravity. (See page 22.)

Of the many forms of apparatus
adapted to the optical methods, one of
the best is the lactoscope devised by
Feser (Fig. 5). A wide glass tube con-
tains in the constricted lower end a milk-
glass cylinder, the wall of which is 4.75
mm. from the surrounding tube. At
regular intervals on this cylinder, sev-
eral equally heavy black bands are
glazed. The surrounding tube is pro-
vided with a double scale; the left grad-
uated from o to 200 and the right from o
to 10. For the determination the tube
is filled with the milk sample to the zero
mark. Water is then added until after
shaking the black lines of the milk glass
cylinder just become visible. The per-
centage of fats is indicated directly by
the right hand scale at the surface of the
liquid.

By the Marchand-Tollens’ Lacto-
butyrometer (Fig. 6) results are ob-
tained more easily but are not so ac-

curate.
Ten c.c. of milk are _ placed

in the narrow glass _ tube,

DETERMINATION OF FATS. 41

acidified with acetic acid and shaken with ro
c.c. of ether. Then adding 10 c. c. of alcohol
(96 per cent), the tube is placed in a water
bath at 4o degrees and allowed to remain for
a short time. The fats dissolve in the ether
and collect on the surface. On the graduated
20 |J scale, the length of the layer of fat solution is
read off, and from this figure the percentage
of fats in the sample is determined from the
tables which accompany the apparatus. In
large dairies and creameries a similar method
al el for fat estimations: is used almost exclusively.
A measured quantity of the milk is treated
with a special acid solution, acetic acid or
sulphuric acid (for Laval’s lactocrite) in a
graduated tube, which is placed in a centrifu-
gal machine and revolved at the rate of six

30

OMT ()
li h ea

Fic. 6. thousand to seven thousand turns per minute.
The By this means the fat separates out as a clear
Marchand-

Tolless compact layer. According to Laval, Ger-
Lactobuty-

rometer. ber, Demichel, Rose- Gottlieb, and oth-

ers, these processes, making use of centrifugal force, give
results that are very useful in commercial practice. They
are especially well adapted to work in large creameries
because a large number of tests may be made in a short
time.*

*The Babcock Method.—One of the most expeditious methods
for ascertaining the percentage of fats in milk is the so-called
Babcock test. It is a simplified modification of the lactocrite
method described above. The results obtained by the process are
very satisfactory, and usually accord well with the results of
ether extractions.

In brief the method consists in measuring out, by means of a
special pipette, 17.6 c. c. of the milk sample and transferring to
a graduated Babcock flask. To this is added 17.5 c. c. of strong
sulphuric acid (sp. gr. 1.81 — 1.83). The milk and acid are thor-
oughly mixed by gently rotating the flask. By this treatment the
casein dissolves completely forming a dark brown fluid, and the

42 METHODS OF QUANTITATIVE ANALYSIS.

IV. ESTIMATION OF ALBUMINOID SUBSTANCES.

A. DETERMINATION OF TOTAL NITROGEN. (Ac
cording to Kjeldahl.)

Ten c. c. of milk are placed in a round bottom flask c
hard refractory glass, and treated with 15 c. c. of pur
cone. sulphuric acid. The acid should be added grad
ually and mixed thoroughly by shaking. To this is adde
one c. c. of metallic mercury or 0.5 gram of mercuri
oxide. The flask is supported in an inclined position o:
a wire gauze, and gently heated, at first with a sma
flame. Gradually the heat is increased until the content
of the flask begin to boil. This temperature is then main

calcium salts precipitate out as insoluble sulphate. The heat c
the reaction with the acid is sufficient to melt the fats, and t
keep them in a molten condition during the next step of th
process. While still hot the flasks are placed in a centrifugz
machine and whirled for about five minutes. The speed of th
centrifugal should be maintained at 1,000 to 1,200 revolution
per minute. After rotating for five minutes, boiling water :
added till the column of fats comes entirely within the range
graduations on the neck of the flask. The flasks are then quick]
returned to the centrifugal and revolved again for one or tw
minutes. In this way the molten fats are collected in a clear]
defined column in the graduated stem of the flask. By subtrac
ing the number on the scale at the bottom of the column fro)
the number at the top, the percentage of butter fats is show
directly. The extreme points of the meniscus at the top and :
the bottom are to be considered as the terminals of the colum:
The reading should be taken as quickly as possible and befo1
the fats solidify.

Experience has shown that in this process, the strength of tl
sulphuric acid used must be approximately as stated. If the ac:
be too strong it will carbonize the lactose and particles of carbc
will rise to the surface of the liquid. On the contrary, if the ac
be too dilute, the casein will not be entirely dissolved and part «
the unchanged curd will appear on the surface. In either case tl
line of demarkation of the fat column will not be sharply d
fined.

For determining the per cent of fats in skimmed milk and
cream, special forms of flasks are used for each. In the case |
skimmed milk twice the usual quantity of sample is taken, ar
the reading obtained is divided by two.

For a more complete description of this method see Wiley
“Principles and Practice of Agricultural Analysis,” Vol. II
page 499.—Translators.

DETERMINATION OF TOTAL NITROGEN. 43

tained for an hour and a half. In this way a clear yellowish
or colorless solution is obtained which contains all of the
nitrogen of the milk in the form of ammonium sulphate.
The flask used in this process should be long-necked
(about 15 cm.) and should have a capacity of 150 c. ¢.
to 200 c. c. Care should be taken in digesting the sam-
ple with sulphuric acid. Sudden heating is liable to cause
bumping and spurting and consequent loss of material.
Heating on a sand*bath is not to be recommended, be-
_cause the flask will.more easily check or crack and be-
cause the temperature is difficult to regulate. Instead of
mercury, 0.5 gram of anhydrous copper sulphate may be
used although, according to Munk, it is necessary in such
cases to digest with sulphuric acid for 20 hours, in order
to completely convert the nitrogenous matter into am-
monium sulphate.* When mercury is used the transfor-
mation may be effected in one to one and a half hours.
Many analysts recommend heating the substance with
sulphuric acid and sulphuric anhydride, or with sulphuric
acid and phosphoric anhydride, or with sulphuric acid and
potassium bichromate. Numerous experiments have
shown, however, that for the determination of nitrogen
in milk, pure sulphuric acid is entirely satisfactory.
When the digestion with acid is complete the con-
tents of the flask are cooled, and transferred to a liter
distillation flask. The small flask is rinsed several times
with distilled water in order to remove every trace of
sulphtric acid. To prevent bumping while distilling add
a few fragments of pumice stone. Then add Io c. c. of
a 50 per cent solution of potassium sulphide to decompose
xercuro-amides (see note below), and finally enough
pure sodium hydrate solution to produce a pronounced
alkaline reaction.

*If copper sulphate be used, the addition of potassium sul-
phate later is unnecessary.

44 METHODS OF QUANTITATIVE ANALYSIS.

A piece of litmus paper may be thrown in for an in-
dicator. When the strong allali is being added the flask
should be immersed in cold water to prevent the solution
from becoming hot and consequent loss of ammonia.
The distilling flask is now connected with a Liebig con-
denser and a 150 c. c. to 200 c. c. distilled off. The dis-
tiilate which contains all of the ammonia, is collected in
a flask containing a measured quantity of standard acid.
In order to prevent any of the boiling fluid from being
carried over mechanically, a Stutzer-Rietmeyer or Koe-
nig safety tube should be used for connecting with the
condenser.

Instead of distilling, the ammonia may be driven out
by forcing steam through the solution. For this pur-
pose the flask is fitted with a two-hole stopper. Through
one hole is put a short safety connecting tube and
through the other, a long tube which reaches nearly to
the bottom of the flask. This latter tube is connected
with a steam generator.

For collecting the distillate and for absorbing the am-
monia, a so-called Pelligot tube or a simpler and cheaper
Erlenmeyer flask of 500 c. c. capacity may be used. Into
the absorption tube or flask 25 c. c. of a fifth normal solu-
tion of sulphuric acid are carefully measured and treated
with a few drops of Congo-red, rosolic acid or cochineal
solution, for an indicator. Twenty-five c. c. of acid is
sufficient for to c. c. of milk. The ammonia in the distil-
late is conducted through a condenser and then into the
acid.

Sulphuric acid is most satisfactory as an absorption
fluid for the ammonia. It is easily titrated and has the
advantage that it does not change in strength by long
standing. For an indicator any of the following solu-
tions may be employed: Congo-red, one gram in
one liter of water: with ammonia produces a red-brown

‘

DETERMINATION OF TOTAL NITROGEN. 45

color. Cochineal, three grams in 50 c. c. of alcohol and
200 c. c. of water: alkalies, especially ammonia, turn it
violet. Rosolic acid, two grams in one liter of 50 per cent
alcohol, gives with alkalines and ammonia a cherry-red
color. Litmus is not applicable in this analysis. The
residual sulphuric acid, not neutralized by the ammonia
distillate, is titrated with fifth-normal potassium hydrate.
From the number of cubic centimeters of sulpuhuric acid
used, that is, the number neutralized by the ammonia, the
nitrogen content of the milk sample may be calculated.
One c. c. of fifth-normal sulphuric acid is equivalent to
0.0034 grams of ammonia or to 0.0028 grams of nitrogen.

From the total nitrogen found, the amount of album-
inoid nitrogen may be calculated by multiplying by 0.94
for cow’s milk, or by 0.91 for human milk. Since the
albuminoids of the latter (casein, globulin, and albumin)
contain 15.76 per cent of nitrogen, the percentage of this
material is determined by multiplying the per cent of
albuminoid nitrogen by 6.34. For cow’s milk the mul-
tiple 6.37 is used (Munk).*

In order to make a large number of determinations by
this method at the same time, a variety of pieces of appa-
ratus have been devised (Hefter, Holbrung and Morgen,
Kreusler, and others). A common form of apparatus
for the purpose is described below. The digestion rack
for the treatment of the samples with sulphuric acid is
made as follows: a circular sheet-iron plate supported
on standards serves for the platform. Into this are cut
six round holes arranged in a circle. Each hole is cov-
ered with a wire gauze which is depressed slightly to fur-
nish a receptacle for the round bottom flasks. In the
center of the plate is placed a small iron standard which

*Compare the details of these methods with those of the off-
cial methods of the Association of Official Agricultural Chem-

ists. Bulletin No. 46, U. S. Dept. of Agr., Division of Chemis-
try, page I4 et seq.—Translators.

46 METHODS OF QUANTITATIVE ANALYSIS.

supports a smaller sheet-iron plate. Around the edge of
this plate six semi-circular notches are cut, equally dis-
tant from one another. The necks of the flasks fit into
these notches and are thus supported in an oblique po-
sition. For the heating, six Bunsen burners are fitted at
proper intervals to a gas pipe bent in the form of a circle.
Each burner is controlled by a stopcock. For the dis-
tillations a simple iron stand with sheet iron top is used.
Six holes are cut through and covered with wire gauze
to support the flasks. Bunsen burners fixed to a main
gas pipe supply the heat. The condenser consists of a
large rectangular tank, supported on a suitable stand and
provided with an inlet and outlet for cold water.
Through the tank are put six wide glass condensing
tubes, the connections being made with tightly fitting
rubber stoppers.

B. METHODS OF PRECIPITATION OF ALBUMIN-
OIDS.

In technical analyses and market control tests, where
it is unnecessary to determine the exact distribution of
the nitrogen in various combinations, the method of
Kjeldahl’s for the estimation of total nitrogen has
proved most satisfactory and reliable. For the
direct determination of albuminoid bodies a large
number of processes have been proposed. (Alco-
hol precipitation; precipitation with tannin, pre-
cipitation with acetic acid according to Hoeppe-
Seyler, and with copper sulphate and sodium hy-
drate according to Ritthausen.) J. Munk has recently
made a study of these methods and has suggested sev-
eral modifications which tend to simplify the processes
and render them more accurate. Only those methods
which have proved to yield satisfactory results and which
are applicable to all sorts of milk will be considered here.
These methods all depend upon the complete precipita-

DETERMINATION OF ALBUMINOID NITROGEN. 47

tion of the albuminoid bodies, and the determination of
the nitrogen in the precipitate by the Kjeldahl process.
A determination of the nitrogen in the filtrate gives the
so-called nitrogenous extract or non-albuminoids.

In order to precipitate every trace of albuminoids from
the milk sample, any one of the following substances may
be used: Tannin and sodium chloride in the cold (Se-
belien’s process), copper hydroxide in a boiling solution
(Ritthausen-Stutzer), or trichloracetic acid in the cold
(Rondsezinsky).

I. Prectpitation with Tannin.

In a 250 c. c. beaker, 10 c. c. of milk are diluted with
go c. c. of water and treated with about 5 c. c. of a sat-
urated aqueous solution of sodium chloride,* and an
excess of tannin or of Almen’s solution (5 grams tannin ;
5c. ¢. acetic acid, 50 per cent; and 200 c. c. alcohol, 30 to
50 per cent). The resulting precipitate settles quite
quickly from a clear yellow supernatant fluid, free of
albuminoids. The liquid is decanted upon a small Swed-
ish filter and the precipitate washed repeatedly by de-
cantation with distilled water, and finally transferred to
the filter. The precipitate should be washed until the
filtrate shows no reaction for chlorine. Acidify a few
drops of the filtrate with nitric acid and add a drop or
two of silver nitrate, when if no turbidity ensues, the
washing may be considered complete. To entirely re-
move the soluble matter by washing requires considera-
ble time because the precipitate quickly fills the pores of
the paper, allowing the water to percolate but slowly.
This difficulty may be largely overcome by washing the
precipitate thoroughly by decantation. The precipitate,

*In all kinds of milk, and especially in human milk, the addi-
tion of salt is necessary in order to effect a complete precipita-
tion by the tannic acid.

48 METHODS OF QUANTITATIVE ANALYSIS,

collected on the filter, is finally transferred to a Kjeldahl
flask and digested with sulphuric acid in the usual man-
mer.* From this point on the determination is con-
ducted in the same manner as described for total nitro-
gen. From the albuminoid nitrogen content thus ob-
tained, the percentage of albuminoids is calculated by the
formulas given above (see page 45).

2. The Ritthausen Method (Modification proposed ly

Munk).

The albuminoid bodies are precipitated from a boil-
ing solution by Stutzer’s copper hydroxide reagent and
the nitrogen estimated in it by the Kjeldahl method.

The copper hydroxide is prepared in the following
manner: 100 grams of crystallized chemically pure cop-
per sulphate are dissolved in five liters of water and treat-
ed with two and a half grams of glycerine. Dilute sodium
hydrate is then added which precipitates the copper as
cupric hydroxide. The precipitate is then filtered off
and washed with water containing 0.5 per cent glycer-
ine by decantation until the filtrate is free of alkali.
The filtrate must be entirely colorless indicating the
absence of copper. If it be still blue more sodium
hydrate must be added. The filtered mass is then
treated with water containing 10 per cent of glycer-
ine and so diluted that it may be readily drawn up into
a pipette. The mixture should be kept in a tightly stop-
pered blue or brown bottle. The proportion of copper
hydroxide per cubic centimeter may be determined by
drying a measured volume of the material and weighing
the residue. Before using this reagent it should be
shaken vigorously in order to mix the precipitate uni-
formly.
~ *The nitrogen content of Swedish filters is usually small but

should be determined in a “blank” and proper deduction made
in the calculation.

DETERMINATION OF ALBUMENOID NITROGEN. 49

Previous to the precipitation of the albuminoid sub-
stances with Stutzer’s reagent the milk must be treated
with alum solution to decompose the alkaline phos-
phates contained. Otherwise in the presence of cupric
hydroxide these phosphates suffer decomposition in
which copper phosphate and free alkali are formed. This
latter will then dissolve the albuminoids and render the
estimation inexact. By the addition of alum this objec-
tionable reaction of the alkaline phosphates is prevented
owing to the formation of alkaline sulphates and insolu-
ble aluminum phosphates.

Tor the analysis by this process 10 c. c. of milk are
placed in a 250 c. c. beaker and diluted with 100 c¢. c.
water (for human milk 60 c. c. water are used). The mix-
ture is then heated nearly to boiling, 2 c. c. of saturated
alum solution are added and when the fluid begins to
boil 5 c. c. of Stutzer’s reagent are run in and the mix-
ture kept at the boiling point for five minutes. The re-
sulting precipitate which settles quickly is washed with
hot water until the filtrate shows no reaction for sul-
phuric acid (the addition of barium chloride to a few
drops of the filtrate acidified with hydrochloric acid
should produce no turbidity). The filter and contents
are then placed in a Kjeldahl flask and treated in the
usual manner.

The reaction of the diluted milk should be noticed be-
fore adding the copper reagent. If it be alkaline it should
be exactly neutralized by the careful addition of alum
since free alkali as mentioned above, dissolves the al-
buminoids and consequently prevents the precipitation
by the copper hydroxide. From the nitrogen content
thus determined the percentage of albuminoids may be
calculated according to the formula given.

3. Precipitation with Triohloracetic acid (Bondsczin-

sky).

50 METHODS OF QUANTITATIVE ANALYSIS.

Ten c. c. of milk are diluted with 50 c. c. of water
and treated with Io c. c. of a 15 per cent. aqueous solu-
tion of trichloracetic acid. This causes a complete pre-
cipitation of the albuminoids. The reaction takes place
in the cold. The precipitate is allowed to stand a few
hours and then washed with dilute trichloracetic acid
and finally the nitrogen content of the residue deter-
mined by the Kjeldahl method.

VII. DETERMINATION OF LACTOSE.

For the estimation of lactose any one of the following

methods may be used. First—Titration with Fehling’s
~ solution. Second—Gravimetric Analysis by the Soxhlet-
Allihn method. Third—Determination by circular po-
larization.

A. TITRATION WITH FEHLING’S SOLUTION.

Before titrating with Fehling’s solution the milk sam-
ple must be freed of albuminoids. For this purpose the
filtrate obtained from the precipitation of the albumin-
oids by the Munk-Ritthausen method may be used (page
48). This filtrate, together with all of the wash water
is well mixed and made up to a definite volume (usually
200 c. c.). An aliquot portion of this solution is then
titrated with Fehling’s solution of known reducing pow-
er. This reagent is prepared in the following manner. It
consists of two solutions which should be kept separate.

Solution 1. 34.639 grams pure crystallized copper sul-
phate are dissolved in warm water and the solution after
cooling made up to exactly 500 c. ¢.

Solution 2. 173 grams of pure crystallized Siegnette
salts (potassium sodium tartrate) and 50 grams chemic-
ally pure sodium hydrate are dissolved separately in dis-
tilled water and the cooled solutions then united and made
up to 500 ¢. c.

DETERMINATION OF LACTOSE. 51

For use in the analysis equal volumes of solutions one
and two are mixed together. Since the mixture of the
two solutions suffers decomposition by standing, they
should be kept separate until required for use. They
should be kept in well stoppered bottles.

The following are the details of the process: Io c. c.
of each of the two reagents are mixed in a small porcelain
evaporator, diluted with 50 c. c. of water and heated to
boiling. Into the solution a few cubic centimeters of the
filtrate containing the sugar are run in and heated for a
few moments. It is then allowed to stand till the red
cuprous oxide settles out. The color of the supernatant
liquid is then noted. If it be still blue a few more c. c. of
the sugar solution are run in from the burette and again
heated and allowed to settle. This process is continued
until the solution becomes colorless. Another sample of
the Fehling’s solution is then measured out and titrated
with the sugar solution as before. The previous deter-
mination will serve as a guide so that nearly the required
amount of sugar solution may be run in at once. The
exact end point may then be determined by careful titra-
tion drop by drop. At least three tests of this sort should
be made. The first observation is usually discarded.

The exact point of complete discoloration of the fluid
is often difficult to detect because of the incomplete sep-
aration of the precipitate. The use of side tests of potas-
sium ferrocyanide for the determination of the end re-
action, as formerly recommended, is considered unsatis-
factory. The solution should not be allowed to stand too
long in order to permit the precipitate to settle com-
pletely because the cuprous oxide easily oxidizes to cu-
pric oxide by the action of the oxygen of the air and dis-
solves again in the alkaline solution producing a blue
color. If the end reaction be difficult to determine with
precision, a small sample of the solution may be filtered

52 METHODS OF QUANTITATIVE ANALYSIS.

out and the filtrate placed in a small test tube and held
over a white paper. In this way any blue coloration may
be readily detected. In case the solution be still blue this
filtered portion may be returned to the evaporator and
titrated further with the sugar solution. If the filtrate
shows a yellowish tint it is probable that too much of the
sugar-bearing filtrate had been used.

The amount of filtrate required to reduce 20 c. c. of
Fehling solution is equivalent to 0.134 gram of lactose.
Upon this fact, the calculations are based. For example,
if one uses 10 c. c. of milk and separates out the fats and
the albuminoids and makes up the filtrate to 200 c. c.
and finds that 50 c. c. of it are required to reduce 20 c. c.
of Fehling’s solution, then the percentage of milk sugar
contained in the milk will be calculated by the formula
4 X 0.134 x 10, which is equivalent 5.36 per cent.

Where great accuracy is not required this method of
titration is useful because results may be quickly ob-
tained. It is especially well adapted for technical analy-
ses, for analyses of food stuffs, and for clinical tests. In
cases where a greater degree of accuracy is demanded
gravimetric methods of analysis should be used. For
standardizing and controlling the Fehling’s solution a
solution of chemically pure milk sugar or dextrose is
used. In the latter case one-tenth of a gram will reduce
20 c. c. of Fehling’s solution.

B. SOXHELT AND ALLIHN’S GRAVIMETRIC
METHODS.

In estimating the lactose by these methods also, the al-
buminoids and fats of the milk must be eliminated. For
this purpose the modified Ritthausen method is employed.
The filtrate is collected in a 200 c. c. flask and the volume
completed with water. By using 10 c. c. of milk and
making to 200 c. c. the most favorable concentration is

DETERMINATION OF LACTOSE. 53

obtained. For conducting the reduction according to this
method of Soxhlet’s, 100 c. c. of the filtrate is added to 50
c. c. of Fehling’s solution, prepared by the formula given
above, and boiled for about ten minutes. At the end of
this time the reduction is complete and the cuprous oxide
which separates out is filtered off. Owing to the diffi-
culty of filtering out this precipitate completely, several
processes of filtration have been devised (Soxhlet, Al-
lihn, Pfeiffer). The filtration may be carried on easily
and successfully by using a hardened filter (Schleicher
and Schull). The filter is fitted tightly into the funnel
and saturated with hot water and the hot supernatant
liquid cautiously decanted, care being taken to retain the
precipitate in the beaker. This precipitate is washed
thoroughly with hot water by decantation. The soluble
matter in the paper is completely removed by washing
with water and finally the precipitate is transferred to the
filter and again washed to remove the last traces of sol-
uble copper. By careful manipulation the precipitated
cuprous oxide in the majority of cases will be completely
retained by the filter. If, however, any of the precipitate
should run through it is best to begin the determination
again. It is important in every case to remove all traces
of the excess of Fehling’s solution from the filter before
transferring the precipitate. The washing of the cuprous
oxide in the beaker should be continued until the wash-
ings show no alkaline reaction or give, when acidulated,
no test for copper with potassium ferrocyanide. Soxhlet
and Allihn suggest the use of a bulbed tube provided with
an asbestos filter for carrying on these filtrations.

The precipitate on the filter is dried at 100 degrees and
ignited in a porcelain. crucible previously dried and
weighed. Continue the ignition until the filter is entirely
incinerated. The flame is then removed and the copper
oxide covered with a thick layer of powdered sulphur.

54 METHODS OF QUANTITATIVE ANALYSIS.

The cover, provided with a gas inlet tube (Rose crucible),
is then put on and hydrogen gas passed in until all of the
air is displaced. Finally ignite for fifteen minutes in an
atmosphere of hydrogen. By this means the copper ox-
ide is converted into copper sulphide and is weighed as
such. From this weight the known weight of filter ash
is deducted and the percentage of lactose calculated from
the accompanying table.

Instead of converting the cuprous oxide into copper
sulphide it may be reduced to metallic copper by heating
in an atmosphere of hydrogen and weighing the resulting
copper. Or instead of this the precipitated cuprous ox-
ide may be collected on a filter previously dried and
weighed and dried at 100 degrees to constant weight.
The increase in weight of the filter will indicate the amount
of cuprous oxide. A still more simple process is to ignite
the filter and contents in a crucible as described above
and moisten with a few drops of conc. nitric acid. The
acid is then carefully evaporated off and the copper ni-
trate which is formed decomposed by ignition. From the
weight of black cupric oxide thus obtained the amount of
milk sugar may be calculated. For very exact determina-
tion the cuprous oxide should always be ignited in an
atmosphere of pure hydrogen and weighed either as
copper sulphide or metallic copper. For the calcula-
tion, find that number in the accompanying table which
is nearest to but less than the number for the copper
sulphide or metallic copper found. Subtract this from
the number found and multiply the remainder by the
corresponding factor, as given, and add to the result the
number for lactose given in the table. For example as-
sume the weight of metallic copper obtained to be 0.2046
grams. The number in the table nearest to and less than
this is 0.2040 grams. The lactose equivalent is 0.150
grams and the corresponding factor is 0.78. Then from

TABLES FOR THE CALCULATION OF LACTOSE.

Copper sulphide|F actor for 1 mg.) Metallic copper |Factor for 1 mg.
found of copper found of metallic Lactose
sulphide copper

0.1733 0.60 0.1383 0.76 0.100
0.1816 0.60 0.1449 0.76 0.105
0.1899 0.60 0.1515 0.76 0.110
0.1981 0.60 0.1581 0.76 0.115
0.2064 0.60 0.1648 0.76 0.120
0.2147 0.61 0.1714 0.77 O15
0.2229 0.61 0.1779 0.77 0.130
0.2308 0.61 0.1844 0.77 0.135
0.3390 0.61 0.1910 0.77 0.140
0.2472 0.61 0.1975 0.77 0.145
0.2553 0.62 0.2040 0.78 0.150
0.5634 0.62 0.2106 0.78 0.155
0.2715 0.62 0.2171 0.78 0.160
0.2795 0.62 0.2235 0.78 0.165
0.2876 0.62 0.2300 0.78 0.170
0.2957 0.62 0.2364 0.78 0.175
0.3037 0.62 0.2428 0.78 0.180
0.3118 0.62 0.2493 0.78 0.185
0.3119 0.62 0.2557 0.78 0.190
0.3280 0.62 0.2621 078 O.195
0.3360 0.62 0.2686 0.78 0.200
0.3441 0.62 0.2750 0.78 0.205
0.3522 0.62 0.2815 0.78 0.210
0.3602 0.62 0.2879 0.78 On2dS
0.3693 0.62 0.2944 0.78 0.220
0.3764 0.62 0.3008 078 - 05225
0.3844 0.62 0.3072 0.78 0.230
0.3925 0.62 0.3137 0.78 0.235
0.4005 0.62 0.3201 0.7 0.240
0.4087 0.62 0.3266 0.78 0.245
0.41607 0.62 0.3330 0.73 0.250
0.4248 0.65 0.3391 0.82 0.255
0.4324 0.65 0.3452 0.82 | 0.260

* 0.4401 0.65 0.3513 0.82 0.265
0.4477 0.65 0.3575 0.82 0.270
0.4554 0.68 0.3636 0.86 OL 229)
0.4627 0.68 0.3694 0.86 0.280
0.4700 0.68 0.3752 0.86 0.285
0.4773 0.68 0.3811 0.86 0.290
0.4846 0.68 0.3869 0.86 0,295
0.4919 0.68 0.3921 0.86 0, 300

DETERMINATION OF LACTOSE. 55

this the amount of copper found represents (0.2046—
0.2040)x0.78-+0. 150=0.0006x0.78+0,150=0.1505 grams
of lactose. In case the precipitate is weighed as cuprous
oxide (Cu,O) or a cupric oxide (CuO), the following ad-
ditional data is required to complete the calculation: One
gram of cuprous oxide represents 0.8882 gr. of metallic

copper and I gram of cupric oxide represents 0.7989
grams of copper.*

C, ESTIMATION BY MEANS OF CIRCULAR POLARI-
ZATION.

In a flask of about 150 c. c. capacity are placed 50 c. c.
of milk; 25 c. c. of neutral lead acetate are added, thor-
oughly ‘mixed by shaking, and heated to boiling. In or-
der to prevent the loss of water by evaporation, the flask
is connected by means of a cork to a long glass tube
which serves as a condenser. After heating for a short
time, the contents of the flask are cooled to room tem-
perature and filtered through a dry filter. The filtrate
should be perfectly clear, otherwise it must be filtered
again. In some cases it may be necessary to filter several
times. With the clear filtrate fill an observation tube
200 mm. long, taking care to force out all the air bub-
bles and determine the angle of rotation in a suitable
polarization apparatus. The specific rotatory power of
lactose at 20 degrees is + 52.53 circular degrees, for so-
dium light. If the angle be ( » ), and if the tibe used be
200 mm. (2 x 100) long, then the number of grammes of
lactose per 100 c. c. of milk will be calculated by the

formula
(a): _100_ 2 aes nx) 1.4277
52.53 2 2
*Compare these methods .with the methods adopted by the
dooce aainon of Official Agricultural Chemists. See Bulletin No.
46 U. S. Dept. of Agr., Division of Chemistry, page 40-41,—
Translators,

56 METHODS OF QUANTITATIVE ANALYSIS.

The polarization may be made in a Landolt’s half
shadow instrument. The observation tube should first be
filled with distilled water, put in place in the polariscope
and a series of readings taken to establish the zero point:
that is, the point at which both halves of the field are
equally illumined. When this point is fixed exactly, the
tube is filled with the filtrate to be examined, and the
polariscope reading is made. The field is adjusted until
the light on both sides is again of equal intensity. Sev-
eral readings should be made in order to insure greater
accuracy.

VIII. DETERMINATION OF CITRIC ACID.

In the ordinary commercial analyses the estimation
of this acid may be omitted, since, from the amount of
it present, no conclusions as to the quality of the milk
may be drawn. To isolate the acid, a large quantity
(about 20 liters) of the milk is used. This is first skimmed
and then allowed to coagulate. The serum is clarified
by heating with acetic acid, and then filtered. To the
filtrate lead acetate solution is added. The resulting pre-
cipitate is filtered off and washed and suspended in
water and decomposed with hydrogen sulphide. Thelead
sulphide formed is filtered off and the filtrate evaporated.
The residue is cooled, and extracted with ether. The
ether solution is then placed in a small weighed dish and
evaporated to dryness on a water bath. The residual
citric acid is finally dried over concentrated sulphuric
acid in a desiccator and weighed. (Vaudin’s Method.)

The volumetric method for citric acid, proposed by A.
Scheibe, depends upon the precipitation of the acid by
means of alcoholic ammonia solution, and the titration of
the triammonium citrate with chromic acid. The latter
is reduced to chromium sesquioxide and the citric acid

*One litre of cow milkcontains 0.9 to 1.0 grams of citric acid in
the form of calcium citrate. Henkel, Jahresber, u die Fortsch,
der Thierchem., 1888, 94; 1891, 129; Schiebe, ibid 1891, 130.—
Translators.

DETERMINATION OF CITRIC ACID. 57

salt oxidized at the same time to carbon dioxide and
water.

A solution of ferrous sulphate is used to indicate the
end reaction. At the moment when all the citric acid is
decomposed, the excess of chromic acid oxidizes the fer-
rous salt to the ferric condition, the change being recog-
nized by the action of potassium ferricyanide (ferric salts
give no precipitate with potassium ferricyanide, while
ferrous compounds give a blue precipitate). The pro-
cess is carried out in the following manner:

“400 c. c. of milk* are mixed with 4 c. c. of sulphuric
acid (2% times normal strength) and heated to boiling;
10 grams of kaolin are mixed with enough water to form
a thin paste and added to the above. The mixture is
again boiled and then allowed to cool. When cold it is
diluted to 500 c. c. and filtered. If the filtrate is not clear
it is again treated with kaolin. To 100 c. c. of the fil-
trate add enough barium hydroxide solution to com-
pletely precipitate the sulphuric acid present, and evap-
orate to a syrup. This residue, which should be stirred
to render as homogeneous as possible, is treated with
2.2 c. c. of sulphuric acid (2!4 normal) and 20 ¢. c. of
absolute alcohol, and after allowing to stand for a short
time is mixed with 50 c. c. of ether. The mixture is then
filtered through cotton. In this way the lactose which
settles out as a crystalline precipitate, is completely sepa-
rated. The filtrate is now treated with alcoholic am-
monia, the reagent being added as long as any turbidness
is produced in the solution. The ether is distilled off,
leaving about 20 c. c. of residue. This is treated with
60 ¢. c. of absolute alcohol and 10 ¢. ¢. of alcoholic am-
monia. By this means the citric acid separates out as
the triammonium salt. On standing a well crystallized

*From Hoppe & Seyler’s “Handbuch der Physiologisch—und
Pathologisch—Chemischen Analyse,” VI. page 471.

58 METHODS OF QUANTITATIVE ANALYSIS.

precipitate is formed which contains all of the citric acid
together with some ammonium chloride ‘and a little or-
ganic matter. This latter may be separated by repeating
the precipitation. ‘

“The ammonium citrate is dissolved i in water and con-
centrated to about 20 c. c. and titrated with standard
potassium bichromate. A solution containing 46.1 grams
K,Cr.O, in a liter, is titrated against a solution of ferrous
ammonium sulphate, prepared by dissolving 150 gr. of
the salt in 700 c. c. water, adding 100 c. c. of concen-
trated sulphuric acid and completing the volume to one
liter; 20 c. c. of this iron solution diluted with 80 c. c.
of water requires 7.7 to 7.8 c. c. of bichromate solution
for complete oxidation. For the titration of citric acid,
the 20 c. c. of this citrate solution are treated with 20-30
c. c. of bichromate solution and 20-25 c. c. of concen-
trated sulphuric acid and carefully mixed by stirring.
After heating for about a quarter of an hour the oxida-
tion to carbonic acid may be considered complete (the
solution should not be heated to boiling). The solution
is then diluted with 50 c. c. of water and treated with an
excess of ferrous ammonium sulphate until the reddish
brown color is completely changed to green. With the
standard bichromate the solution is titrated back until no
more ferrous salts can be detected with potassium ferri-
cyanide. Theoretically 4.61 grams of potassium bichro-
mate are equivalent to I gram of citric acid. Numerous
titrations have shown, however, that in practice 4.61
grams K,Cr,O, represent 1.02 grams of citric acid. The
lactic acid contained in the milk does not affect the re-
sults because this acid is not precipitated by alcoholic am-
monia.””*

*See also “Zeitschift fur Analytische Chemie,” 1899, page
718; also “Analyst,” 23, page 161; and also “Revue Chim. Analyt.
Appl.” VI (7) 110. —Translators.

DETECTION OF PRESERVATIVES.

In order to prevent milk from souring and to deceive
the customer as to the freshness of it, a number of differ-
ent preparations are made use of as preservatives. In
some cases these preservatives are used for the purpose of
preventing the curdling of the milk samples which are to
be subjected to analysis, and which for example have to
be sent a long distance during hot summer months. To
such a procedure no objection could be raised. If, how-
ever, milk thus treated be offered for sale as food, vigor-
ous protestations should be made against it from a hy-
gienic standpoint. Such milk should be rejected as food
and by all means as food for infants. All assertions and
opinions to the contrary are prejudicial and unreliable.

The objection to the use of preservatives is based on
the fact that they hinder or prevent coagulation. On this
account it is impossible to judge of the age of the milk or
to decide whether it is fit for use. Concerning the value
of various preparations commonly used as preservatives,
it may be said that sodium carbonate and sodium bicar-
bonate are not especially active as germicides. In fact,
many of the pathogenic micro-organisms such as cholera
bacilli, develop only in alkaline culture-media. Borax
and boric acid act slightly as bacteriacides, while forma-
lin and salicylic acid act strongly. The latter, however,
is entirely ineffective against typhus bacilli. Benzoic
acid and hydrogen peroxide are effective only when used
in large quantities.

In practice the following are quite extensively used:
borax and boric acid, sodium carbonate, salicylic acid,
benzoic acid, formalin, chromium—, mercury—, and am-
monium salts, and sodium fluoride. The last four are
used only for analytical work. The first are found most
frequently in milk as sold by the unscrupulous purveyor.

59

60 DETECTION OF PRESERVATIVES.

For this reason improved methods for detecting these
substances are given below.

I. BORAX AND BORIC ACID.

’

For qualitative tests about 100 c. c. of milk are made
alkaline with milk of lime, evaporated to dryness and in-
cinerated. The resulting carbon need not be completely
burned. The heating is stopped as soon as intumescence
ceases. The ash is dissolved in the least possible amount
of hydrochloric acid; filtered off from the carbon; evap-
orated to dryness; and the excess of HCl completely
driven off. The resulting white crystalline residue is
treated with a few drops of a tincture of tumeric and very
dilute hydrochloric acid and dried on a water bath. The
presence of the slightest trace of boric acid gives to the
dry residue a beautiful vermillion or cherry red color
(Meissel). By the described method it is possible to de-
tect with certainty 0.001 to 0.0005 gram of boric acid in
the ash or 0.001 to 0.002 per cent in the milk.

To render the milk alkaline, lime water is preferable
to the caustic alkalies because, in fusing, the latter are
liable to cover over particles of unburned organic matter
and make the conversion to ash difficult. Only very dilute
hydrochloric acid must be used in the final evaporation
of the crystalline residue in testing for boric acid since
the concentrated acid itself gives with tumeric tincture a
red color. The coloration produced by boric acid is dis-
tinguished from that produced by hydrochloric acid by
the fact that it does not disappear by treatment with water
in the cold, but only after long boiling. The color caused
by hydrochloric acid disappears as soon as it is diluted
with water. The acid is easily soluble in alcohol and a
trace of it in alcoholic solution colors the flame of the
Bunsen burner an intense green. With alcohol, boric

BORIC ACID AND BICARBONATE. 61

acid ester is formed. If the alcohol be ignited a flame
tinged with green is produced.

For the quantitative ‘determination of boric acid Cassal
proposes the following process: 100 grams of milk are
made alkaline with sodium hydrate, evaporated and in-
cinerated. The ash is washed with water and methyl
alcohol into an Erlenmeyer flask fitted with a two hole
stopper. Through one of the holes is placed an adapter
projecting a short distance into the neck of the flask and
connected with a condenser. Through the other hole
is placed a separatory funnel filled with methyl alcohol.
The contents of the flask are acidified with acetic acid
and placed in an oil bath and subjected to distillation. The
distillation product is collected in a platinum crucible
filled with pure quicklime. This is placed in a large glass
beaker which is covered with a glass plate with a hole in
the center. Before using, the crucible and contents
should be ignited and weighed. Through the perforation
in the plate a bent glass tube connected with the free
end of the condenser is passed, allowing it to extend
down to the crucible. The distillation is repeated about
ten times, shaking each time with 5 c. c. of methyl alcohol.
The crucible is finally dried and heated strongly with a
blast. The increase in weight gives the amount of boric
acid in 100 c. c. of milk.

II. SODIUM CARBONATE, SODIUM BICARBONATE.

Ten c. c. of milk are mixed with 10 c. c. of alcohol (96
per cent) and a drop of rosolic acid solution (1 :100).
Pure unadulterated milk produces a brownish-yellow
color, but in the presence of sodium carbonate or sodium
bicarbonate a rose color is obtained. For greater pre-
cision in doubtful cases the questionable sample should be

62 DETECTION OF PRESERVATIVES.

compared with known unadulterated milk. Phenolph-
thalein solution may not be used, as an indicator in place
of rosolic acid in this test. By the method given 0.05 per
cent of these carbonates may be easily detected (Hilger,
E. Schmidt).

III. SALICYLIC ACID.

(a) Acidify 20 c. c. of milk with two or three drops of
sulphuric acid and shake with about an equal amount
of ether. The greatest possible part of the ether solu-
tion is drawn off and evaporated; the residue extracted
with 40 per cent alcohol; filtered; and 5 c. c. of the fil-
trate treated with a few drops of ferric chloride. A violet
color shows the presence of salicylic acid. By comparison
with a solution of salicylic acid of known strength and
colored by treatment with ferric chloride the approximate
amount of acid present may be estimated. (Remont.)

(b) 100 c. c. of milk are diluted with 100 c. c. of water
at 60 degrees and treated with eight drops. of mercuric
nitrate, thoroughly shaken, and the resulting precipitate
filtered off. The filtrate which will contain all of the sali-
cylic acid originally in the milk sample is shaken with
50 c. c. of ether; the ether solution is drawn off, filtered
through a dry filter and allowed to evaporate spontane-
ously in the air. If the residue resulting be white and
crystalline, soluble in alcohol and produces a violet
color with a few drops of one per cent ferric chloride so-
lution the presence of salicylic acid is proved conclu-
sively. (Girard.)

IV. BENZOIC ACID.

A large quantity of milk (300-500 c. c.) is made alkaline
with calcium or barium hydroxide solution; evaporated
to one-fourth its volume; mixed with pure sea sand,
pulverized pumice stone or gypsum, to a thick paste and

DETECTION OF BENZOIC ACID. 63

evaporated to dryness on a water bath. The mass is then
finely pulverized, moistened with dilute sulphuric acid
and shaken with twice its volume of cold fifty per cent
alcohol. The alcohol dissolves out milk sugar, salts, and
benzoic acid; only a trace of fats dissolves. The solution
is neutralized with barium hydrate and without filtering
off the barium sulphate which separates out, evaporated
to small volume (10 c. c.) and again acidified with
dilute sulphuric acid and extracted three or four times
with a small amount of ether. By evaporation of the ex-
tracts pure solid benzoic acid remains. (The residue is
usually mixed with a trace of fats.)

For a quantitative determination the above residue is
dried at 60 degrees or over concentrated sulphuric acid,
weighed, and then heated upon a water bath until the ben-
zoic acid sublimes off. In order to identify the benzoic
acid subsequently, the sublimate should be collected.
This may be accomplished by inverting an evaporator
over the other dish. Finally the dish and residue is
weighed back and the loss of weight, that is, the differ-
ence between the two weighings, indicates the weight of
benzoic acid.

The presence of benzoic acid may be confirmed by sub-
jecting the sublimate to the following tests: (1) A small
amount is heated gently in a watch glass, over which is
inverted another glass of the same size. The acid, if pres-
ent, sublimes and condenses on the upper watch glass
in fine white glistening flakes. (2) A small sample is
treated with a few drops of fuming nitric acid, and evap-
orated to dryness. The dry residue is mixed with sand
and heated strongly in a glass ignition tube. In conse-
quence of the formation of nitrobenzol a strong odor of
bitter almond oil will be noticed.

64 DETECTION OF PRESERVATIVES.
V. FORMALIN.

This substance, which has now come into general use
as a food preservative, according to some authorities,
may be properly used as a preservative for milk. The
commercial preparation “Formalin” consists of a 40 per
cent solution of formaldehyde in water. The detection
of it in milk is a simple matter.* By adding a solution
of silver nitrite to the milk a black precipitate is formed
when formaldehyde is present. In case of very small
amounts of formalin, the nitrite solution produces usual-
ly, on standing, only a brownish black stain or coloration,
due to the production of finely divided metallic silver.
The preservative action of formalin is especially marked.
It has been found that milk may be kept 100 hours at 25
degrees C. when formalin is added in the proportion of
one part of formaldeyde to 5,000 parts of milk. When
added in such small amounts, formalin changes neither
the odor nor the taste of milk. Notwithstanding this, its
use as a preservative of milk intended for food should be
prohibited since it has been shown that even with dilute
solutions it retards digestion and also alters the compo-
sition of the milk. Upon the albuminoids especially it
exerts an influence tending to increase the solubility of

*For the detection of formaldehyde in milk a method has re-
cently been proposed making use of phloroglucin solution. 0.1
gram of phloroglucin is dissolved in 100 c. c. of water. I-2c. c.
of this solution is treated with a few drops of potassium or sodi-
um hydroxide and added to eight or ten c. c. of the milk to be
tested. In the presence of formaldehyde a red coloration is pro-
duced. According to Jorrissen one part in 20,000 may be easily
detected in this way. This reaction with phloroglucin takes
place only with dilute formaldehyde solutions. Vanino found
that ten to thirty per cent solutions gave no reaction, or at best
only a slight coloration. A three per cent solution produces a
deep red color. The strongest color is produced by 0.5 per cent.
With solutions containing 0.00004 per cent formaldehyde the
color is distinctly visible but when more highly diluted it does
not respond distinctly to the phloroglucin test. See Zeitsch. fur
Anal. Chemie, 1900, page 64.—Translators.

CHROMIC ACIDS, MERCURY SALTS. 65

these in acetic and sulphuric acids. The results of quan-
titative analyses by the methods given are unreliable
when formalin is present, and hence it is a question if this
is the best material to be used even in preserving milk
samples that are to be kept some time before being an-
alyzed. (Thompson, Merkel, Kruger.)*

VI. CHROMIC ACID AND OTHER PRESERVATIVES.

Chromic acid, potassium bichromate, as well as am-
monium salts and mercury salts, and ammonia, all serve
as preservatives of milk samples, but for obvious reasons
are used only in samples to be analyzed; 0.25 gram of
potassium bichromate is sufficient to prevent the coagu-
lation of 250 c. c. of milk for two months. Chromic acid
is used as a 20 per cent aqueous solution. One drop of
this in 100 c. c. of milk will keep the latter fresh for eight
days; 25 per cent ammonia produces a similar effect.
The preserving power of any of these substances is large-
ly dependent upon the temperature of the milk. In no
case should it be allowed to rise above 10 degrees C. The
results of comparative tests before and after treatment
with these preservatives have shown that in the deter-
mination of fats, etc., these substances do not interfere.

**Experience of late years in laboratery and anatomical work

has abundantly shown that formaldehyde ——— hardens al-
buminous matter and converts it into a substance with a leathery
AP PCATANGEs. se acagewyainan ¢ Gottstein (Deut. Med. Wach, XXII.

797) determined that such food products when hardened by the
use of formaldehyde cannot again be softened by boiling.”—Dr.
Young, Secretary of State Board of Health of Maine.

The Cornell Experiment Station bulletin No. 118 states: “The
behavior in the Babcock test of milk which has been preserved
hy formalin shows that its composition is in some way affected.
ere crete When formalin is used the curd often fails to dis-
solve (in sulphuric acid) and becomes a hard compact mass.”

For the effect of formaldehyde on the proteids of milk see also
Annalen der Chemie. Vol. 310. p. 25 (1800); Zeitsch. fur Physiol.
Chem., 22, 127 (1800); Vermont Experiment Station 11th An-
nual Report, page 333.—Translators.

66 DETECTION OF PRESERVATIVES.

VII. SODIUM FLUORIDE.

A one per cent solution of this salt will prevent the
souring of milk from three to five days. This strength
of solution is usually employed. No practical method has
been suggested for detecting this substance.

DETECTION OF ADULTERATIONS.

I. ADULTERATION WITH WATER.

By far the most common adulterant used for milk is
water. This adulteration cannot always be detected by
ordinary market control tests since, as already stated, the
effect of the water which is added is often counteracted
by skimming the milk. The result is frequently in such
cases that the specific gravity is not essentially different
from that of the normal original sample. Sometimes it
is possible to discover such admixture by means of chem-
ical reactions. Such tests are based on the fact that in
almost every case the water used as a diluent contains
considerable quantities of salts such as calcium sulphate
and other sulphates and often traces of nitrates which are
foreign to pure milk.

Pure milk contains only an exceedingly small amount
of sulphuric acid, never more than 0.3 per cent (calculated
to ash). In case a considerably larger amount is found,
the addition of water is indicated. The test is conducted
in the following manner: 50 c. c. of milk are treated with
acetic acid to precipitate the casein, filtered, and the fil-
trate evaporated, dried, and incinerated. The ash is taken
up with hot water and a few drops of hydrochloric acid;
and the extract filtered, boiled, and treated with barium
chloride solution. After heating for some time the re-
sulting barium sulphate is filtered off and washed first
with hot dilute hydrochloric acid and then with boiling
water to remove the acid. The filter and contents are
dried, placed in a weighed crucible, ignited, and weighed.

67

68 DETECTION OF ADULTERATIONS.

By subtracting the weight of the filter ash, the weight of
the barium sulphate is obtained. One part BaSQ, is
equivalent to 0.3429 part SO .

For the detection of nitric acid a satisfactory method is
to coagulate the milk by means of a solution of calcium
chloride free of nitrates, filter off the precipitate, and mix
with the filtrate a solution of diphenylamine in concentra-
ted sulphuric acid. In the presence of nitric acid, even of
minute traces of it, there appears a blue ring or zone at
the line of demarkation of the two liquids.

Egger and Moslinger recommend a solution of 0.02
gram diphenylamine in 20 c. c. of sulphuric acid (1 part
H:SO. and 3 parts H:O) completing the volume to 100
c. c. with concentrated sulphuric acid. Of this solution
2c. c. are placed in a porcelain evaporator and to it 0.5
c. c. of the milk serum is added. The serum is obtained
by heating 100 c. c. of milk with 1.5 c. c. of concentrated
solution of calcium chloride and filtering from the result-
ing precipitate. After adding the serum the material is
allowed to stand for a moment or two, then mixed by
rotating the dish, again allowed to stand and again mixed.
In the presence of only traces of nitric acid a blue streak
develops around the edge of the dish and by mixing grad-
ually colors the whole fluid.

Both of these methods for detecting adulteration fail
if distilled water containing neither sulphates nor ni-
trates is used as a diluent. A number of other methods
for this purpose have been proposed but they give only
approximate results. Some of them, however, are of
value to many workers, physicians for example, who of-
ten lack time and apparatus necessary for a more exact
test. If by one or more of these simple methods, results
are obtained which are considered doubtful, as will occa-
sionally happen, the sample may be submitted to a chem-
ist for a more refined chemical analysis.

ADULTERATION WITH WATER. 69

For the above reason a few of the best processes are
here outlined. These apply only to cow’s milk.

(1) The method proposed by Lezé and Hilsont is to
determine the time required to produce coagulation in a
known quantity of milk by the action of rennet. This
method gives at the same time a means of judging some-
_what as to the freshness of the sample. Upon unadul-
terated fresh milk at 35 degrees C., rennet reacts in forty
minutes when added in the proportion of 1:10,000, that
is, one liter of rennet will cause ten thousand liters of
milk to curdle in forty minutes when the temperature is
35 degrees. In this process a solution of good commer-
cial rennet is used, one c. c. of it being added to 100 ¢. ¢.
of milk. © :

With the above proportions (1:100), pure milk coagu-
lates in three minutes and eleven seconds. When 10 per
cent of water is added it requires three minutes and four-
teen seconds, and with 50 per cent of water, five minutes
and forty-nine seconds. If the time of coagulation ex-
ceeds 3 min. 50 seconds, the addition of water or some
alkaline preservative is evidenced. On the other hand, if
the milk coagulates in less than two minutes it evidently
was not fresh, the sudden curdling being due to incipient
decomposition. Such milk is unfit for use as food.

(2) For this purpose of detecting the admixture of
water to milk, Lescoeur suggests coagulating the sample
with rennet and determining the density of the serum
at 15 degrees C., and also the total dry matter contained
in it. The density of serum of pure milk varies from
1.029 to 1.031, and the dry matter per liter varies from
67 to 71 grams. The addition of four per cent of water
diminishes the density about 0.001, and the dry matter
about two grams per liter. In making comparisons the
following table may be used:

70 DETECTION OF ADULTERATIONS.

Density | ‘Total dry mat-
Condition of the Sample. at1s°c ter per liter.

Pure mille sscanbsesawryaces ces 1.0300] 70.0 grams
100 parts milk + 10 parts water../1.0275| 64.0 “

100 parts milk + 20 parts water. .|1.0251 59.0
100 parts milk + 30 parts water. .| 1.0230 54.5

“cc

“cc

(3) By Beckman’s method the freezing point of the
milk is determined. The results are not influenced by the
fats present, but are dependent upon the water content.
Normal milk freezes at — 0.554 degrees C. (average).
The lowering of the freezing point below that of water
is proportional to the concentration. It is lowered about
one-half the above number by diluting the milk with an
equal volume of water. The sample in question is
brought to the freezing point by means of a mixture of
ice and salt. A similar test is carried on with a sample
of distilled water. The difference in the observed freez-
ing points indicates the quality of the milk. An addition
of 10 per cent of water produces a diminution of only
0.055 degrees.

From the results of a large number of analyses an
average normal number has been established for the
amount of each of the constituents of milk. By com-
paring other analyses with these figures adulteration may
be often exposed. For example, if the fat content of the
dried matter in milk is found to be less than 27.6 per cent
and the specific gravity of the same less than 1.335, there
is but little doubt of falsification (Herz). The nitrogen
content of unadulterated milk should never be less than
0.5 per cent ;* the ash never less than 0.7 per cent. The
relation of ash to the dry matter, minus fat, varies from

*Results of 15,000 analyses.

ADULTERATION WITH STARCH. 71

1:8 to 1:8.5 (Droop-Richmond). Further, in normal
milk the ratio of casein to fat should never be greater
than 1 :1.74 and never less than 1:1.35.* (L. Van Slyke.)
Unfortunately it must be admitted that these normal
numbers as given will not cover every case without ex-
ception. The composition of cow’s milk (other kinds of
milk need not be considered here) is influenced by so
many factors such as climate, breed, feeding, keeping,
duration of lactation, etc., so that universal normals can-
not be established absolutely.

A never failing proof still remains, namely, the “stall
test.” In cases of extreme importance a number of sam-
ples of milk may be obtained directly from the animals in
the stalls. An analysis of these samples furnish data with
which results of tests of suspected milk may be compared.
Since the fat content and consequently the specific grav-
ity is subject to considerable variation in different sam-
ples, much more reliance is put upon the determination
of the other constituents, viz., albuminoids, ash, sugar.

II. ADULTERATION WITH STARCH, DEXTRIN, ETC.

Sometimes it is found that skimmed milk is treated
with starch in the attempt to restore the original white
color. The detection of this adulterant by chemical means
is a simple matter.

By acidifying with acetic acid and boiling, the milk is
freed from albuminoids. The filtrate obtained is treated
with a few drops of a dilute solution of iodine. In the
presence of starch the well known blue color appears.
This test, however, is no longer to be considered as proof
positive of starch. There is in milk and milk products
another substance giving a similar reaction. This sub-
stance is considered by Herz as an amyloid on account
of its similarity to the amyloid discovered by Virchow

*Results of 25,931 analyses.

72 DETECTION OF ADULTERATIONS.

in pathologic spleen, liver and kidneys. Microscopic
tests show that amyloid is similar to starch in size, form
and behavior. There is, however, a sharp distinction be-
tween them in their reactions with water. By heating
with water amyloid does not form a paste such as starch
forms. For proving the presence of starch the results of
microscopical examinations must be confirmed at least
by the iodine and water tests. The same holds good for
the detection of dextrin, glue, or gums, sometimes used
for adulteration. For these, too much reliance should
not be placed upon the results of tests by any one
method.

ESTIMATION OF INSOLUBLE FOREIGN
MATTER.

RENCK’S METHOD.

A liter of milk is placed in a high, cylindrical vessel
and allowed to stand at least two hours, after which time
the greater part is removed either by decanting or
siphoning. To the rest containing the suspended parti-
cles of solid matter, water is added to make up the orig-
inal volume. This is allowed to stand again for two
hours, siphoned, and the process continued until the
fluid in the vessel is almost clear and no trace of milk is
left. The liquid is then carefully siphoned off leaving only
a small amount, without disturbing the sediment. The
sediment is then carefully filtered through a filter which
has been dried at 105 degrees and weighed. It is washed
several times with distilled water, then, to remove any
particles of fat contained in the sediment, it is washed
with alcohol and ‘ether, and then dried at 105 degrees to
constant weight, and weighed. The difference in weight
between the clean filter paper and that containing the
sediment gives directly the amount of foreign matter in
one liter of milk. According to Renck, the milk sold in
Berlin averages 10 milligrams to the liter.

In order to remove the dirt and estimate the amount
contained in milk bottles used by dairies in delivering

ol
63

74 ESTIMATION OF INSOLUBLE MATTER.

milk, a very simple method is recommended by Stutzer:
A heavy test tube (c, Fig. 7) is fastened air tight by
means of a piece of rubber tubing (b) to the mouth of a
milk bottle, (a). The bottle is inverted and allowed to re-

main in this position for some time. The particles of in-
soluble matter settle in the bottom and collect in (c).
“The connection between the test tube and bottle is then
shut off by a clamp, the test tube removed, and the con-
tents treated according to the method described above.

EXAMINATION OF CONDENSED MILK AND
CURDLED MILK.

I. CONDENSED MILK.

The examination of condensed milk is made accord-
ing to the methods described for ordinary milk. A
stated amount of the condensed preparation is diluted
with a given amount of water and the previously de-
scribed methods employed. The estimation of dry mat-
ter obviously is made with undiluted milk.

To prove the presence of diluted condensed milk in
fresh milk use is made of the fact that a part of the sugar
is caramelized in evaporation, thereby becoming optically
inactive. Therefore the estimation of the quantity of
sugar by use of the polariscope gives lower results than
the volumetric or gravimetric methods of analysis. Usu-
ally the difference between the two methods in fresh milk
is never more than 0.15 per cent. (Droop-Richmond.)

II. CURDLED MILK.

Curdled samples are examined according to the usual
methods. For the determination of fat the curd is dis-
solved by adding a few drops of a dilute solution of po-
tassium or sodium hydrate. The specific gravity is best
ascertained by a method of Weigul’s as follows:

A definite volume of the milk (Vm) is mixed with a
known volume of ammonia (Va) of known specific grav-
ity. By shaking, the curd dissolves completely. The
volume of the solution (\s) is equal to Vm + Va. The
specific gravity of the solution (Ds) is determined by
means of an accurate areometer. From the data thus
obtained the specific gravity (Dm) of the curdled milk

may be calculated by the formula: Dm—Ssx Ds Vax de

75

BACTERIOLOGICAL EXAMINATION OF MILK.

7

I? INTRODUCTION. METHODS OF PREPARATION.
CULTIVATION.

Milk generally contains a large amount of micro-or-
ganisms, the number of which varies according to the
cleanliness practised in the milking and in the entire man-
agement of the dairy. It has been demonstrated that
the number of germs present depends upon the amount
of foreign matter contained in the milk. From the mo-
ment the milk leaves the udder, the bacteria increase
very rapidly provided their vitality is not decreased or
weakened by heating or by the use of preservatives, so
that by estimating the number, an idea can frequently be
gained as to the freshness of the milk, as well as the
cleanliness of the process by which it is handled.
However, bacteria may be contained in the milk even
before it leaves the udder, which is the case in certain
diseases, the diagnosis of which can be made through
a bacteriological examination of the milk. In human
milk, which was drawn from a mammary gland, and kept
scrupulously clean and under perfectly aseptic conditions
by every precaution, various cocci have been found
(Honingmann, Lewis, Polleske, Johannessen, Durante,
and others).

In examining bacteria of milk the simple dry
cover-glass preparation is sufficient in many cases,
but the fat must always be extracted. <A _ bit
of the milk to be examined is taken up on a loup made
at the end of a platinum wire and spread with water upon

76

QUANTITATIVE ESTIMATION OF GERMS, 77

a clean cover-glass that is free of all grease, forming a
film as thin and even as possible. It is dried without
the use of a flame. The cover-glass is then placed in
ether for a few minutes, by which process the fat is ex-
tracted. It is then taken out and stained in the usual
manner. C. Arens combines the two processes of fat-
extraction and staining by placing the cover-glass prepa-
ration in a chloroform-methyleneblue solution, of the
following composition: 12-15 drops of a saturated alco-
holic solution of methyleneblue and 4 c. c. of chloro-
form. It is stained from 4 to 6 minutes and washed.

This simple method is not always successful. The
various modifications will be discussed below.

Il. QUANTITATIVE ESTIMATION OF GERMS.

For estimating the number of germs contained in milk
the gelatine-plate method is employed, using either Koch
or Petri dishes. After a stated time the developed colo-
nies are counted with the aid of a counting apparatus.
Wolffhugl’s counting apparatus is a very convenient de-
vice where the Koch plates are employed. .\s milk usu-
ally contains a great number of germs the sample must
be properly diluted; more in summer than in winter. Of
milk, not rich in germs, I ¢. ¢. is diluted with 9 c. c. of
sterilized water and 1c. ¢., equal to 0.1 of this mixture, is
then inoculated into to c. c. of nutrient gelatine. It 1s
customary to estimate the number in 1 c.c. The estima-
tion of the number of bacteria may serve as an indication
of the quality of the so-called sterile milk 1 commerce.
Milk is seldom rendered germ-free, for by so doing seri-
ous chemical changes very often take place effecting the
appearance, taste. agreeableness and digestibility.

When the plate culture method is used in isolating
and growing the different bacteria contained in the milk,
dilutions of the original inoculated tubes must be made
in the usual way.

78 BACTERIOLOGICAL EXAMINATION OF MILK.

Ill. MILK ANOMALIES.

The so-called disease of milk, or ‘milk anomalies,”
often have their origin in the action of the presence of
micro-organisms. The most important of these anoma-
lies are the following:

I. fed Milk. This is caused by different sapro-
phytic (non-pathogenic) genii of bacteria: Sarcina spe-
cies, Bacillus prodigiosus, Bacillus lactis erythrogenus,
Bacillus rubidus, Spirillum rubrum, Micrococcus cinna-
bareus, and by a red yeast.

(a) Of the Sarcina, the most common are Sarcina
rosea Menge, Sarcina rosea Schrotter and Sarcina au-
rantiaca. The different species, of which numerous germs
are found in the air, are distinguished from one another
by their readiness or slowness in liquefying gelatine.
Aurantiaca does not liquefy gelatine, and grows very
slowly. All grow upon the ordinary culture media and
are strongly aerobic.

(b) The Bacillus prodigiosus is a very small, short
rod, possessing no motion. It grows as well at room
temperature as at 37 degrees and liquefies gelatine. At
incubation temperature there is no pigmentation, the
latter condition going hand in hand with the production
of trimethylamine (odor like that of pickled herring).

A dark red color is beautifully shown upon potato
culture; this becomes light red when treated with acetic
acid. Ammonia restores the original deep red color.
Sterile milk inoculated with Bacillus prodigiosus slowly
precipitates the casein; the serum-zone beneath the layer
of cream gradually becoming blood red.

(c) Bacillus lactis erythrogenus (Hueppe) liquefies
gelatine. In bouillon and solid culture media it pro-
duces a yellow pigment, while in milk and whey the
pigment produced is red. The red color is impaired by
light and acids.

MILK ANOMALIES. 79

(d) The Bacillus ruber is a long, thread-like, very
motile rod, which when isolated is grouped in threes or
fours and occasionally shows from two to four spores.
Upon agar a yellow layer is formed. It liquefies gela-
tine. Potato cultures are covered with a rusty red film.

(e) Spirillum rubrum is a thick, clearly transparent
bacterium, having a regular spiral movement and is
provided at each end with a flagellum. The long spirilla
exhibit slow motion while the short ones are actively
motile. Reproduction takes place by division. Spore
formation is doubtful. The spirillum grows between 16
degrees and 4o degrees, best at 37 degrees. Upon gela-
tine the growth is very slow, non-liquefying. In plate
cultures many small, grayish-red colonies the size of a
pin head are formed. In stick cultures, round, reddish
granular colonies are formed along the track of the
needle. Upon agar and blood serum a grayish white,
sharply defined layer is first developed, later producing
thick, rosy-red layers. Upon potato a moist glistening
layer, reddish in color, is formed.

(f) Micrococcus cinnabareus is a large coccus often in
the form of a diplococcus and often grouped in threes and
fours, and does not liquefy gelatine. It grows very
slowly. Upon. plates, after about four days, the deep
colonies appear as pale brick colored points, In gelatine
stick cultures, white colonies are seen along the line of
punctures and upon the surface rose-colored buttons
develop, becoming dark red in time.

(g) Sometimes the red color of the milk is due to col-
oring matter contained in the fodder or blood.

2. Yellow Milk, Yellow milk is caused by the Bacil-
lus synxanthus (or Bacterium synxanthus Ehrenburg).
The micro-organism develops as an actively motile small
rod, which, when inoculated into sterilized milk, gives it
a citron yellow color. The yellow coloring matter disap-

80 BACTERIOLOGICAL EXAMINATION OF MILK.

pears under the action of acids and is restored by neu-
tralizing the acid with an alkali. It is insoluble in alcohol
and ether. Yellow milk has a sickening odor and taste.

3. Blue Milk. The Bacillus cyanogenus, Bacillus
cyaneofluorescens Zangemeister, and Bacillus janthinus,
all impart a blue color to the milk.

(a) The Bacillus cyanogenus or Bacillus: syncyaneus
is a small rod frequently grouped in twos. Numerous
flagella on the sides give it a vigorous motion. The bacil-
lus grows readily on slightly acid media, best at room
temperature. Gelatine plate cultures show superficial
colonies and stick cultures exhibit dark gray layers. It
grows well on media containing grape sugar and glycer-
ine. Sterilized milk becomes slightly alkaline and blue
gray. Unsterilized milk becomes dark blue. This bacillus
does not otherwise tend to decompose the milk. . Its ac-
tivity being dependent upon the formation of pigments.

(b) Bacillus cyaneofluorescens Zangemeister differ
very sharply from the above. It is short and oval in
shape, motile, does not liquefy gelatine, but gives it a
yellowish green fluorescence. Upon sugar gelatine it
grows as a white film. The cultures have a strong odor
of trimethylamine. Unlike Bacillus cyanogenus, it
does not effect sterilized milk, but gives it a blue color
when inoculated together with the Bacillus acidi lactis.

(c) Bacillus janthinus, a rod, grows on gelatine plates
as a white film, gradually becoming violet at the border.
The stick culture is violet only on the upper surface. On
potato a violet colored layer is formed. Sterilized milk
shows blue spots on the cream-surface. After a short
time the casein is precipitated, an alkaline reaction is
shown and an abundant amount of ammonia is formed.

4. Stringy Milk, The micrococcii of Schmidt-Muhl-
heim give milk a slimy property, capable of being drawn

MILK ANOMALIES. 81

out into threads. The bacteria of Duclaux and bacteria
lactis viscosis Adametz do likewise. The latter flourishes
on all media, especially well upon peptonized glycerine
gelatine. It causes sterilized milk to become threadlike
after three or four weeks. The Micrococcus viscosus of
Schmidt-Muhlheim grows in garland-chains and pro-
duces a slime similar to plant-slime. Contrary to many
other slimy fermentation processes caused by micro- or-
ganisms, no carbonic acid is found. More definite data
concerning the cultivation of the Schmidt-Muhlheim
coccus is lacking.

5. Slimy Milk. Micrococcus viscosus, a bacillus
found by Weigmann and Zirn and by G. Leichmann,
does not render milk ropy, but slimy and soapy.

(a) Micrococcus viscosus grows in chains and gives
sterile milk a gummy, slimy viscosity.

(b) Weigmann isolated from slimy, soapy milk, four
species of bacteria, one of which gives to sterilized milk
the above mentioned peculiarities, while the others mere-
ly produce a yellow coloring. The species in question
produces a slimy precipitate and an alkaline reaction,
the precipitate disappearing after a short time, the milk
becoming watery and fluorescent.

(c) Leichmann’s bacillus is a slender rod with rounded
corners, single or in pairs, seldom lying in chains, which
decomposes sterilized milk up to 50 degrees, developing
considerable gas and causes the whey to become slimy.

6. Bitter Milk. Proteus vulgaris Hauser, Bacillus
von Bleisch, Bacillus and Micrococcus liquefaciens lactis
amari Freudenreich, the bacteria of bitter mill of
Hueppe, Flugge (as below), also a number of bacteria
found by von Sterling, produce bitter milk. The bitter
taste is the result of the formation of peptones,
which is found in the serum of the peptonized
milk. By treating the same with potassium or

82 BACTERIOLOGICAL EXAMINATION OF MILK.

sodium hydrate -and a few drops of diluted copper sul-
phate solution, it produces a violet to a rose-red color, the
reaction for peptones (biuret reaction).

So-called ‘‘pozson milk,” which acts like alkaloids
upon animal organisms, is probably due to the presence
of ptomaines or toxins produced by bacteria from the
albuminoids of the milk.

To obviate the above described milk anomalies, which
are mostly the results of insufficient cleanliness, the stalls
must be thoroughly cleaned with water, the walls fre-
quently white-washed, all utensils boiled, the udder care-
fully washed with warm boiled water and the persons en-
trusted with the milking must exercise perfect cleanli-
ness.

Of the numerous micro-organisms contained in nearly
all milk, the peptonizing species, the so-called bacteria
of bitter milk, “Hueppe’s” demand special attention. To
this class belong a number of strongly anaerobic and 12
facultative anaerobic species, recently described by
Fluegge. Under these, according to Fluegge, probably
come Bacillus mesentericus fuscus and vulgaris, Bacillus
liodermis, Bacillus albus lactis Loeffler, Bacillus butyri-
cus Hueppe and Botkin, the bacteria of Duclaux, the
Kreuger bacillus, Proteus vulgaris, Micrococcus Conn,
Clostridium foetidum and butyricum, Bacillus mus-
coides Liborius, and four varieties called by Ster-
ling, bacterium lactis peptonaus. Fluegge has re-
cently published the results of a study of the
bacteria which peptonize milk, or rather the casein
of milk and which for the largest part belong
to the group of hay and potato bacilli. He has also called
attention to their dangerous character. These bacteria are
characterized by forming spores which have extraordi-
nary powers of resistance and which are killed only when
exposed for two hours to the influence of steam at 100 de-

MILK ANOMALIES. 83

grees C. and which therefore retain their full vitality in
milk sterilized according to the usual method. Moreover,
their presence is unnoticed in fresh milk kept at ordinary
temperature or in boiled milk ; their influence, however, is
noticed when imperfectly sterilized milk is kept at 37 de-
grees in an incubator. Such milk, or sterilized milk in-
oculated with pure cultures of peptonizing bacteria,
shows profound changes after standing from one
to five days. Beneath the cream zone a_trans-
parent zone is formed which appears to be made
up only of serum. Gradually the transparent zone
becomes wider, the casein not yet peptonized be-
gins to separate into flakes, and in most cases
rennet fermentation occurs in addition. Milk so
changed, has a bitter, pungent, peptone-like taste. The
identification of individual specics presents great diffi-
culty. It is not possible to recognize them through cul-
tures upon agar and gelatine plates, but identification is
usually successful through cultivation upon potatoes, ab-
solutely sterile skimmed milk, alkaline ¢clatine, and stich
culture in sugar agar. (Often these methods fail and it is
necessary to study the metabolic assimilation and the
thermal death point of the germ to assure an accurate
diagnosis.

(An accurate and detailed description of all the characteristics

of the bacteria mentioned above is found in “Fluegge's Zeit-
schrift | fuer Hygiene and Infectionskrankheiten, Vol. NVII,

292."")

Abnormal milk with neutral or alkaline reaction fre-
quently becomes curdled, due to the presence and the ac-
tion of the bacteria just mentioned. Normal sour curdling
is caused by a series of widely diffused kinds of bacteria,
viz: the Bacillus acidi lactici Hueppe: Bacterium acidi
lactici Grotenfeld; Bacterium limbatum acidilactici
Marpmann: Bacillus acidi lactici Leichmann; Micro-

84 BACTERIOLOGICAL EXAMINATION OF MILK.

coccus Marpmann; Sphaerococcus Marpmann; and
Streptococcus acidi lactici Grotenfeld. The difference
between the individual species lies chiefly in their physi-
ology. For example, Bacillus acidi lactici Hueppe, pro-
duces lactic acid and carbonic acid, and the Bacillus acidi
lactici Leichmann, lactic acid and ethyl alcohol, etc.

IV. DEMONSTRATION OF INDIVIDUAL PATHO-
GENIC BACTERIA IN MILK.

1. BACILLUS TUBERCULOSIS.

Sometimes the cover-glass preparation is successful in
demonstrating the bacteria. For the preparation of such,
different directions are given.

(a) ‘Method of Ahrens: A drop of milk is placed
upon the cover-glass with a drop of sterilized water.
This is dried in the air and fixed by slightly warming.
The cover-glass is then placed in a mixture of 3 drops
of a concentrated alcoholic solution of fuchsin and 3-4
c. c. of chloroform. After ten minutes it is transferred
to 4-6 c. c. of 96 per cent alcohol, to which has been
added 2-3 drops of diluted sulphuric acid. After wash-
ing in water it is double-stained with an aqueous solu-
tion of methylene blue.

(6) Method of Alessi: A drop of milk is dried
upon the cover-glass by moderate warming and 2-3 drops
of one per cent sodium hydroxide solution added. It is
again warmed until saponification of the fat takes place
(three or four minutes), washed with water and stained
as usual, for example, according to Gabbet; 2 minutes in
cold carbolic fuchsin, 50 seconds in cold sulphuric acid
methylene blue Gabbet, i. e., methylene blue 2 parts, 25
per cent H2SO4, Ioo parts.

(c) Method of Schrank: A drop of diluted
milk is dried in the air on a cover glass. The
casein is fixed by placing the cover-glass on a copper

DETERMINATION OF BACILLUS TUBERCULOSIS. 85

sheet and heating it without producing a brown color. It
is then placed in a dish containing 4 c. c. of ether or
chloroform, shaken until the fluid is evaporated and
stained as usual. If with these processes the results are
negative, the attempt must be repeated with a larger
amount of milk. One hundred grams of milk are mixed
with about 5 c. c. of sodium hydrate and shaken with a
large amount of ether in a separatory funnel until no
more fat goes into the solution. A sample of the ether
evaporated will convince one of this fact. The fat-free
liquid is brought into a funnel shaped glass or a sediment
apparatus and allowed to stand for 24 hours. After this
time.a dry preparation of a sample of the sediment is
made after the manner described.

(d@) Method of Thoerner: 20 c. c. of milk
mixed with 1 c. c. of 50 per cent potassium hydrate are
placed in centrifugal tubes and then placed in boiling
water for about two minutes until the milk becomes yel-
lowish-brown. They are then removed and well shaken,
the mixture treated with 20 c. c. of glacial acetic acid,
again thoroughly shaken, placed in boiling water for
three minutes and centrifugalized 10-20 minutes. The
liquid above the sediment is drawn off and the sediment
with about 40 c. c. of hot water again centrifugalized
for ten minutes, and a cover-glass preparation made
from the sediment in the usual manner.

(ce) Method of Itkewitsch: 20 c. c. of milk
are coagulated with citric acid, and filtered. The residue
on the filter is dissolved in a very dilute aqueous-solu-
tion of sodium phosphate, mixed with about Io c. c. of
ether and shaken for 10-15 minutes. The solution found
beneath the fat zone is drawn off, centrifugalized, and
a preparation of the sediment made on a cover-glass.

The cultivation of the tubercle bacilli from milk is
quite difficult. They grow excluded from air and light

86 BACTERIOLOGICAL EXAMINATION OF MILK.

only upon solid culture media of cattle blood serum
glycerine agar. They grow remarkably slow, and, °
spite of great dilution of the milk, a sample from as
the inoculation is made, the media become very quickly
overgrown by the rapid and luxurious growing
saprophytic bacteria of the milk. For the de-
termination of the tubercle bacilli the solid cul-
ture media are not so well adapted as the fluid
ones; of the latter, especially the Nocard bouil-
lon (nutrient bouillon with 6 per cent glycerine and ,
3 per cent gelatine). A large number of ‘samples are
suitably started and kept for at least three weeks at 37
degrees in an incubator. From the colonies developed
in one manner or another, microscopic preparations are
made and stained in the usual manner. :

More accurate information is gained by experimenting
on living animals to determine the presence of tubercle
bacilli in the milk, or whether their number is sufficient
when the milk is consumed to produce tuberculosis.
Guinea pigs and also rabbits, are fed with the suspected
milk and after 8-10 weeks examined for tuberculosis of
the intestines, mesenteric glands and the liver. Instead
of feeding, intraperitoneal injections can be made. The
sediment secured from centrifugalizing impure milk is
especially adapted for this purpose. The presence of
tuberculosis is diagnosticated by sections of the dead or
slaughtered animals.

2. THE CHOLERA BACILLUS.

The demonstration of it presents great difficulty and
must be done according to the proper methods of exam-
ining water. Neither staining, appearance, nor animal ex-
perimentation are especially characteristic. Its identity
can be confirmed only through cultivation.

For the diagnosis of the cholera bacillus a method

THE TYPHUS BACILLUS, 87

given by Koch may be used. Stock cultures are prepared.
In the meantime the material to be examined is put in an
alkaline 1 per cent peptone solution and kept in an incu-
bator at 37 degrees. Within 6 to 12 hours the cholera
bacilli, if present, will have rapidly increased,
gathered at the upper surface of the fluid, at
times forming a fine film (on account of the great need
of oxygen). From the upper surface of the peptone so-
lution, or in other words, from the thin film, agar and
gelatine cultures are inoculated. After iwelve hours
characteristic growths and reactions appear ; best on agar
at 37 degrees and on gelatine at 22 degrees. Regarding
the latter point a complete text-book of bacteriology
nust be referred to. It may be mentioned that the chol-
era bacilli grow only upon alkaline culture media, and
liquefy blood serum. They give a cholera red indol re-
action and have a remarkable tendency to dry up.

3. THE TYPHUS BACILLUS.

The Typhus bacillus is equally as difficult to demon-
strate in milk as is the exciter of cholera, particularly
since it is easily grown over by species very similar to it
and most always occurring together with it, for in-
stance, by the Bacillus coli communnis.

For the differentiation of these two micro-organisms,
one utilizes, besides the different reactions with their
pure cultures (indol reaction, behavior towards sterile
milk, ete.), a culture process (method given by Elsner)
upon plates of potato gelatine mixed with 1 per cent po-
tassium iodide. Upon this media colonies of Bacillus coli
communnis appear after 24 hours as yellowish white
points, while at this period the Typhus bacillus will not
have grown. After 48 hours the Typhus Bacillus forms
small, bright glistening buttons (knobs), while Bacillus
coli communnis forms large brown spots (specks).

88 BACTERIOLOGICAL EXAMINATION OF MILK.

For the preparation of the Elsner culture medi 500
grams of potatoes are extracted with a liter of water; to
this extract 100 grams of gelatine are added; 10 c. c. of
the potato gelatine is made alkaline with 2.5—3 c. c. of
I-10 normal sodium hydroxide mixed with 1 per cent po-
tassium iodide.

The methods of cultivation of the known species, as
well as the possible occurring pathogenic staphylococcus,
and streptococcus, the streptococcus of infectious indura-
tion or the Streptococcus agalactiae contagiosae—the
cause of chronic and acute mastitis of the cow—the
Staphylococcus and Streptococcus Guillebeau, the bacil-
lus Guillebeau, etc., need no special explanation; they
are governed by the universal technique of bacteriology
which has heretofore been mentioned.°

The Bacillus Guillebeau is a facultative anaerobic
motile germ, gas forming, which does not liquefy gela-
tine. In stick cultures it grows needle-like with finely
pointed iris knobs. It causes sterilized milk to coagu-
late. Upon potatoes white or brown layers are formed.
According to Grame the bacillus does not stain. In
Guinea pigs and rabbits it is not pathogenic. Inoculated
into the udder it produces mastitis.

The Staphylococcus mastitis Guillebeau, according
to Grame, is stainable and shows motility. It is faculta-
tive anaerobic, without gas development, liquefies gela-
tine with the formation of a sediment and causes steril-
ized milk to coagulate. Upon potato a yellowish white
layer is formed. The Streptococcus mastitis sporadicae
Guillebeau is also facultative anaerobic, liquefies gelatine
and thrives only very scantily upon potato. By inocula-
tion it shows no pathogenic peculiarities.

APPENDIX.

EXAMINATION OF POWDERED, MALTED MILK AND
OTHER SOLID FORMS OF PRESERVED MILK.

The examination includes the determination of dry
matter (water), ash, phosphoric acid in the ash, the
detection in the ash of injurious metals entered in pack-
ing, fat, albumin, the amount of soluble matter in water
(extract) and carbohydrates (sugar and starch). An
average sample is used.

1. The dry substance is determined according to the
directions for milk (Page 28). Ten grains of the ma-
terial are usually taken for the test.

2. The usual methods hold good for the ash and phos-
phoric acid. In the ash it is important, when metal boxes
are employed for packing, to test for arsenic, antimony,
lead and tin.

3. Fat: 10 grams are extracted with ether in the
Soxhlet extraction apparatus, the ether fat solution evap-
orated and the remaining fat weighed. (Page 31.)

4. Extract: 25 grams are extracted with hot water
and this repeated until nothing more goes into the solu-
tion. The entire amount, with the aid of a funnel pro-
vided with a perforated base (Witt plate), is filtered
through mull or muslin. The aqueous extract (filtrate)
collected is brought to a stated volume (1 liter as a rule)
cooled off and an aliquot part, about 50 or 100 c. c.
evaporated to dryness upon the water bath in a platinum

89

90 APPENDIX.

(or porcelain) dish at 105 degrees until dried to a con-
stant weight, then weighed and the results reckoned on
the whole fluid mass. —

5. Carbohydrates:. From the solution saved from the
extract determination, a measured amount, about 200
c. c. is mixed with 5 grams of concentrated H2SO4
(or 18 ¢. c. of concentrated hydrochloric acid) and
boiled 5 hours in a flask provided with a reflux conden-
ser.* Thereby all the carbohydrates are inverted, that‘is,
they are transformed into reducing sugars and can be de-
termined according to methods given for milk sugar. The
inverted cooled solution is brought at room temperature
to a stated volume, an aliquot part of the same taken and
treated as for the milk sugar determination given on

page 50.

*Such prolonged heating at such high temperature is usually
inadvisable and unnecessary. The common practice at present
is to heat for 15 minutes at 68°—70° C. See bulletin No. 46, U.
S. Dept. of Agr., Div. of Chem., p. 31.—Translators.

BIBLIOGRAPHY.*

I. BACTERIOLOGY OF MILK.

Bacteria in Milk and Its Products. Storrs Agr’l Exp.
Sta. Bulletin No. 4, by H. W., Conn. (1889).

Bacteriological Examination of Milk. By J. Clauss
(Diss. Wurzburg. 1890, p. 518).

Bacteriology of Normal and Abnormal Milk. By L.
Adametz (Nos. 1-3. Oesterreich. Monatssch. fur
Thierheilkunde. 1890).

The Effect of Centrifugal Action on the Distribution of
Bacteria in Milk. By Scheurlen (Arb. a. d. Kais.
Ges. Amt. No. 7. 1891).

The Micrococcus of Bitter Milk. The Ripening of
Cream. Conn, Storrs’ Exp. Sta. Third Annual Re-
port (1891).

Bacteria in the Dairy. Conn, Storrs’ Exp. Sta. Rept.
for 1891.

Bitter Milk and the Sterilization of Milk by Heating
Under Exclusion of Air. By M. Blisch (Ztg. Hyg. No.
13, p. 81. 1891).

The Sterilization of Milk. (Milch. Ztg. 1893).

The Sterilization of Milk on a Large Scale. (Ztg. Hyg.
No. 13, p. 42.)

The Spread of Contagious Diseases in Milk and Dairy
Products. (Molk Ztg. No. 8, 1893.)

*No effort has been made to make this bibliography complete
or inclusive. Only a few of the more important recent re-
searches, bearing directly upon the subject matter of this book,
are here collated.—Translators.

91

é

92 BIBLIOGRAPHY.

The Danger of Consuming Milk of Sick Cows. By J. F.
Baum. (Arch, Wiessensch. u prak. Tierheilkunde.
Vol. 18, No. 3-4.) ;

Some Bacteriological Work in the Dairy. By L. H.
Pammel. Iowa Sta. Bulletin No. 21.

On Ropy Milk and the Source of Bacteria in Milk. (Jour.
of the American Chem. Soc’y. Vol. 15, No. 6.)

A Review of-Recent Work on Dairy Bacteriology. By
E. W. Allen. (Exp. Sta. Rec. Vol. 5, p. 1042.)

Behavior of Cholera Bacilli in Milk and Cheese. By H.
Weigmann & G. Zirn. (Landw. Wochenbl. Schles.
Holst. Milch. Ztg. 23. 1894. No. 20, pp. 311-313.)

Behavior of Cholera Germs in Milk. By H. Weigmann.
(Milch. Ztg. 23 (1894), No. 31, pp. 491-493.)

Effect of Milk on Cholera Bacilli. By W. Heese (Ztschr.
Hyg., 17 (1894), p. 238; abs. in Chem. Ztg., 18, 1894.)

Dairy Bacteria. U.S. Dept. of Agr., Office of Exp.
Stas. Bulletin No. 25. By H. W. Conn.

Bacteriological Investigation in the Dairy. By G. J.
Leufven. (Rept. Ultuna Agl. Inst., 1894.)

The Source of the Bacteriological Infection and the Re-
lation of the Same to the Keeping Quality of Milk.
Wis. Exp. Sta. Rept. for 1894. By H. L. Russell.

The Bacteriological Contamination of Milk. Ont. Agr.
Exp. Sta. and Coll. Rept. for 1896. By F. C. Har-
rison.

The Persistency of Bacteria in the Milk Ducts of Cow’s
Udder. By G.S. Ward. (Jour. of Appl. Micros.
No. 1, 1898.)

Bacillis Typh. Abdominalis in Milk and Butter By H.
F. Bolley and M. Field. (Backtuper to. Abt. No. 24,
1898.) (Centbl. Bakt. u. Parisit., No. 24, 1898, pp.
881, 887.)

BIBLIOGRAPHY. 93

I. PRESERVATIVES AND ADULTERATIONS, METH-
ODS OF DETECTION.

The Influence of Milk Preservation. By A. W. Stokes,
Analyst. No. 16 (1891).

The Preservation of Milk in Norway with Boric Acid.
Weigmann. (Milch Ztg., 1893.)

The Aeration of Milk. Ind. Exp. Sta. Bulletin No. 44.
By C. S. Plumb (1893).

The Recognition of Foreign Fats in Butter. By W.
Johnstone. (Abs. in Chem. Centbl., 1893.)

Note on the Detection of Adulteration of Fresh Milk
with Diluted Condensed Milk. By D. Richmond,
Analyst. (No. 18, 1893.)

Creaming and Aerating Milk. By H. H. Wing. (N. Y.
Cornell Sta. Rept. for 1892, pp. 113-142.)

Notes on the Detection of Formalin. By H. D. Rich-
mond and L. K. Bosly, Analyst. (No. 20.)

The Preservation of Milk and Cream for Household
Purposes. (The Nursery of the Sick Room.) By R.
Bell. (No. 86, p. 13, 1896.)

The Method of Distinguishing Pasturized and Unpastur-
ized Milk. By V. Storch. (Bericht. des. Veruchs-
laboratoriums der, Kgl. Veterinar—und Lardban-
hochschule. Copenhagen. No. 40, 1898.)

Effect of Formaldehyde of Milk. Analyst, Vol. 20, p.

157.
II. VARIATION IN THE COMPOSITION OF MILK.

The Analysis of Milk of Different Breeds of Dairy Cows.
Mich. Exp. Station, Bulletin No. 68. By G. H.
Witcher. (Feb. 1890.)

The Effect of Food Upon the Quality of Milk. Iowa
Sta. Bulletin No. 14. By J. Wilson (1891).

The Curdling of Milk During Thunder Storms. By T.
O. Lemei. (Abs. Milch Ztg. No. 20, 1891.)

94 ' BIBLIOGRAPHY.

The Effect of the Change of Quarters Upon the Quantity.
and Quality of Milk. Vt. Exp. Sta. Rept. for 1891.
By B. L. Hills.

Abnormal Milk. By R. G. Smith. (Jour. Soc. Chem.
Ind., 13 (1894), No. 6, pp. 613 and 614.)

Conditions Affecting the Consistency of Milk. Wis. Exp.
Sta. Rept. for 1896. By S. M. Back and H. R.
Russell.

IV. DETERMINATION OF FATS.

The Babcock Method of Determining Fat in Milk and
Cream. Conn. Exp. Sta. Bulletin No. 106 (March,
1891).

Method of Determining Milk Fat. Penn Sta. Bulletin No.
12. By B. W. Frear and Glholter (1890).

A New Method for the Estimation of Fat in Milk, Espe-
cially Adapted to Creameries and Cheese Factories.
Wis. Exp. Sta. Bulletin No. 24. By S. M Babcock
(1890).

A Discussion of Cochran’s Method for the Determination
of Fat in Milk for the Use of Dairymen. N. Y. Agr.
Station Bulletin No. 17. By G. S. Caldwell (1890).

A Method for the Determination of Fat in Milk and
Cream. N. Y. State Exp. Sta. Bulletin No. 19. By
P. Collier, (Illustrated)

A New Milk Test. Vt. Exp. Sta, Bulletin No. 21 (1890).

The Importance of Extraction of Paper Coils in Milk
Analysis by the Adams Method. Bulletin No. 16. By
P. Vieth, Analyst (1891).

A Comparison of the Methods of Determining Fat in
Milk. By L. F. Nilson. (Chem. Ztg. No. 15.)

Determination of Fat in Sour Milk. By Mekenberg.
Chem. Ztg. No. 15.)

A New Method of Determining the Amount of Butter

BIBLIOGRAPHY. 95

Fat in Milk. Miss. Exp. Sta. Bulletin No. 21. By
L. G. Paterson (1892).

The Best Milk Tester for the Practical Use of the Farmer
and Dairyman. Colo. Station Bulletin No. 20. By
Walter J. Quick (1892).

The Acid Butyrometric Method of Rapidly Determining
Fat in Milk. (Chem. Ztg. No. 98, 1892.)

The Estimation of Solids and Fat in Milk. By J. B. Kin-
near. (Chem. News, No. 68, 1893.)

The Recognition of Foreign Fats in Butter. By W.
Johnstone. (Abs. Chem. Centbl. 1893).

Remarks on Babcock's Method of Fat Determination.
By F. W. Woll. (Tidskr. norske. Landbr. No. 1

(1894), pp. 285-202.)
V. COMPOSITION.

The Constituents of Milk. Wis. Exp. Sta. Bulletin No.
18. By S. M Babcock (1899).

The Analyses of Milk of Different Breeds of Dairy Cows.
Mich. Exp. Sta. Bulletin No. 68. By R. C. Kedzie
(1890).

The Analysis of Milk. N.Y. Cornell Sta. Bulletin No.
25. By H. Snyder (1890).

Composition of Cream and Butter Milk, and the Loss
of Butter Fat in Churning. Conn. Exp. Sta. Annual
Report (1891).

Analysis of Milk and Other Dairy Products. Miss. Sta.
Rept., 1891.

Composition of Dairy Products, Minn, Sta. Bulletin No.
27. By H. Snyder.

Composition of Milk and Milk Products By H. D. Rich-
mond, Analyst. (Mar., 1893.)

Citric .\cid and Phosphate of Lime in Solution in Milk.
Iiv L. Zaudin (Ann. Inst. Pasteur, 8 (1894). No. 17,
pp. 502-505.)

96 BIBLIOGRAPHY.

‘The Chemical Composition of Cow’s Colostrum. By
L. Vaudin. (Bul. Soc. Chem. Paris, 11-12 (1894), No.
13, pp. 623-625.)
_ Frozen Milk. (Milch Ztg. No. 26, 1897.)
Constituents of Milk. Wiley, 464.
Composition of Milk of Different Mammals. Wiley, 465.
Milk, Skim Milk, and Whey; a Study of Their Compara-
tive Composition and Specific Gravity. By C. B.
Cochran. (Jour. Amer. Chem, Soc., Vol 15, p. 347.)

VI. METHODS OF ANALYSIS.

Investigation of Milk Tests. Ill. Exp. Sta. Bulletin No.
10. By E. H. Farrington (1890).

Determination of Casein in Milk. By Jroux. (Chem.
Centrabl. No. 1, 1891.)

The Determination of Nitrogen by the Kjeldahl-Jodlbam
Method. (Exp. Sta. Record. Vol. 5, p. 462.)

Saponifying Butter Fat by the Leffmann and Beam
Method. Del. Sta. Rept. for 1892. By C. F. Penny.

Contribution to Milk Analysis. By E. Beckmann.
(Milch Ztg. 23 (1894), No. 44, pp. 702-703.)

Milk Analysis. By Lescouer. (Abs. in Chem. Centbl.,
1894, IT., No. 19, pp. 816).

Examination of Composite Samples of Milk. By M. Wei-
bull. (Chem. Ztg. 18 (1894), No. 81, p. 1567, 1568.)

Method of Estimating Acidity of Milk. By M. Schaffer.
(Staz. Sper. Agr. Ital., 26 (1894), pp. 164-167; Abs.
in Jour. Chem. Soc. 67-68, 1895.)

The Determination of Specific Gravity of Clabber Milk.
By Rischloff. (Milch Ztg. No. 48.)

A Method for Determining the Acidity of Milk. By A.
Devarda. (Milch Ztg. 1896.)

Importance of Expelling CO, in Determining the Acidity
in Milk. Bulletin No. 47, Div. of Chem., U. S. Dept.
of Agr. p. 127.

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