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http://www.archive.org/details/cu31924003216821

UNIVERSITY OF WISCONSIN STUDIES

IN SCIENCE
NUMBER 2

PAPERS ON BACTERIOLOGY AND
ALLIED SUBJECTS

BY FORMER STUDENTS OF
HARRY LUMAN RUSSELL

MADISON
1921

UNIVERSITY OF WISCONSIN STUDIES

NUMBER 16 SCIENCE NO. 2
JUNE, 1921 PRICE, ONE DOLLAR

Published bi-monthly by the University of Wisconsin at Madison, Wisconsin
Entered as second class matter August 31, 1919, at the postoffice ht Madi-
son, Wisconsin, under the Act of August 24, 1912. Accepted for mailing

at special rate of postage provided for in section 1103, Act of
October 3, 1917, Authorized September 17, 1918.

No. 1. The Fishes of Lake Valencia, Venezuela, by A. S: Pearse.
52 p. Fifty cents.

No. 2. Papers on Bacteriology and Allied Subjects, by Former
Students of Harry Luman Russell. 200 p. $1.00.

DEAN HARRY LUMAN RUSSELL

UNIVERSITY OF WISCONSIN STUDIES

SCIENCE
NUMBER 2

PAPERS ON BACTERIOLOGY AND
ALLIED SUBJECTS

BY FORMER STUDENTS OF
HARRY LUMAN RUSSELL

MADISON
1921

DEDICATED TO

BACTERIOLOGIST DEAN HARRY LUMAN RUSSELL

BY HIS FORMER STUDENTS ON THE TWENTY-FIFTH
ANNIVERSARY OF HIS
DOCTORATE

CONTENTS

A Review of the Scientific Work of Harry Luman Russell
Together with a List of his Books and Scientifie Pa-
pers.

E.G. Hastings, M.S. ’99 . ;

The Development of City Milk Supply Problems
H. A. Harding, B.S. ’96, M.S. ’98 ‘

The Resistance of Mold Spores to the Action of Sonlight,
John Weinzirl, B.S. 796, M.S. 799, Ph.D. ’06

The Influence of the Plane of Nutrition upon the Per-
centage of Fat in Milk and the Properties of the Fat.
C. H. Eckles, Graduate Student ’96—’97

Characteristics and Distribution of the Colon-Aerogenes
Group.

L. A. Rogers, Graduate Student, ’97—’98

The Identity of American and French Sporotrichosis.
D. J. Davis, B.S. 798 :

The Significance of Yeasts and Oidia in Pasteuived But-
ter.

F.W. Bouska and J. C. evant Graduate Students,
*01-’02

The Action of Certain Bactesa on ihe Nececnons Mate.

rial of Sewage.
EL. G. Birge, B.S. .

The Detection of eee Mill,

W. D. Frost, Ph.D. 03 . F

The Investigation of Drinking Water Supplies
H. A. Whittaker, Ph.G. ’03, B.A. ’06

The Milk Supply of Chieago.

A. L. Amott, B.S.A. 708

The Bacteriology of Ice Cream.
B. W. Hammer, B.S.A. ’08

30

60

131

139

151

164

170

182

FOREWORD

In June 1917 the twenty-fifth anniversary of Dean H. L.
Russell’s doctorate was celebrated by a complimentary break-
fast. As a part of the exercises, it was announced that a
quarter century volume by certain of his students would be
published in his honor. Most of the manuscripts were re-
ceived June 1, 1918, but war-time conditions have delayed the
completion of this task until now. It is hoped, however, that
the unavoidable delay will not lessen the pleasure which the
volume will bring to either the teacher or his students.

H. A. Harpina,

E. G. Hastrnes,

W. D. Frost.
Committee.

A REVIEW OF THE SCIENTIFIC WORK OF
H. L. RUSSELL

E. G. Hastines

It is impossible to present in a few pages an adequate
statement of the work of him in honor of whom this volume
is issued. In most of the fields that engaged his attention,
as a member of the Wisconsin Experiment Station staff, the
work was of a pioneer nature and has served as a foundation
for the work of others who were to follow, making a more
detailed study of the varied fields.

The University of Wisconsin has been fortunate in the
loyalty of its staff and in the cooperation that has existed be-
tween its various departments. The work presented by Dr.
Russell in many of his papers was done in association with
colleagues in other departments of the College of Agriculture.
Chief among these collaborators may be mentioned Dr. 8S.
M. Babcock whose knowledge of the chemistry of the dairy
supplemented and indeed made possible much of the work in
dairy bacteriology, a subject that is as much chemical as
biological in nature.

Men are creatures of circumstances. Their careers are
governed largely by their associations. One of the great
factors in directing the future of the subject of this sketch
was his father, a medical man by education, a scholar by
nature, who realized the value of a university education
and who made possible the years of graduate study. H. L.
Russell was born on March 12, 1866, son of E. Fred Russell
and Lucinda Estella Russell. He entered the University of
Wisconsin in 1884, recciving his bachelor’s degree in 1888.

Another important factor in the determination of the future
career of the student was the presence in the University of
Wisconsin of William Trelease, now in charge of the depart-
ment of botany in the University of Illinois. Dr. Trelease

10 UNIVERSITY OF WISCONSIN STUDIES

had been attracted by the new subject, the new science of
bacteriology, that was developing so rapidly under the
tutelage of Pasteur and Koch. ‘The thesis submitted to
Harvard University in 1884 by Professor Trelease for his
doctorate was entitled Observations on Several Zooglea and
Related Forms. It involved a study of the growth of cer-
tain bacteria on potato. The interest of Professor Trelease
in bacteriology led him to introduce it into his courses in
botany. Dr. Trelease left the University in 1885 and his
work fell to the then professor of zoology, President E. A.
Birge, under whose guidance the student was to receive his
introduction to bacteriology. Two years were spent in grad-
uate study at the University of Wisconsin. During this
period the first paper was prepared. It was entitled Pre-
lininary Observations on the Bacteria of Ice from Lake
Mendota, and was published in 1889.

In those days two laboratories were attracting all students
who wished to become bacteriologists, that of Robert Koch
at the University of Berlin and that of Louis Pasteur in the
Institute which bears his name. The years of 1890-1891 and
1891-1892 were spent in these laboratories and at the Zoological
Station at Naples where the data were collected for the first ex-
tensive paper which was published in the Zeitschrift fiir Hy-
quene in 1892 under the title Untersuchungen tiber im Golf von
Neapel lebende Bakterien. On returning to this country a year
was spent under Dr. William H. Welch of Johns Hopkins
University. By this university the doctor’s degree was
granted in 1893. The thesis submitted for the degree was
entitled Bacteria in their Relation to Vegetable Tissue. It
was published in the Johns [Hopkins Hospital Reports in
1893. <A year was then spent at the University of Chicago
as fellow in biology.

Under the influence of Weigmann in Germany, Storch in
Denmark, and Conn in this country, dairy bacteriology was
rapidly attracting the attention of those interested in this
phase of animal industry. It had long been evident to the
director of the Wisconsin Experiment Station, Professor
Henry, that Wisconsin was destined to become a great dairy

SCIENTIFIC WORK OF HARRY LUMAN RUSSELL 11

state. He had brought to Wisconsin the most prominent and
the most honored dairy investigator of this country, Dr. 8S.
M. Babcock, and had sought in every way to favor the develop-
ment of the dairy industry. It was but natural that these
raen, wide awake to the developments that were to come in
dairying, should at once recognize the importance of this new
phase of bacteriology to the state of Wisconsin. It was again
but natural that in seeking a man to develop this subject, both
from’ the research and instructional points of view, they
should turn for aid to the first student of the University who
had prepared himself for work in this field. In 1893 H. L.
Russell was appointed assistant professor of bacteriology in
the University of Wisconsin. Advancement to full professor
was made in 1897.

The training received in general biology led to the develop-
ment of a course in general bacteriology, in which the general
relations of the bacteria were considered, apart from their
practical relations. This course was given most successfully
for many years. The constant knocking at the door of the
list of required studies by the new phases of agricultural
work that had been introduced from time to time into the
college led to the abandonment of the course and to the
development of a course in which the practical aspects of
bacteriology as related to farming were especially emphasized.
From the laboratory of the University have gone many men
who have had much to do with the development of agricultural
bacteriology in this country.

The Wisconsin College of Agriculture in 1893 was expend-
ing a large portion of its energy in the development of the
Dairy School and in the training of young farmers in the
Short Course in Agriculture. Dean Henry furnished to these
groups of students the best instruction the College could pro-
vide. Thus the instructional work of the bacteriologist was
not limited to the regular university students, but was ex-
tended to both the students in the Dairy School and in the
Short Course in Agriculture. Many hundreds of young men
have returned to their homes with not only some idea of the

12 UNIVERSITY OF WISCONSIN STUDIES

great importance of the bacteria in the life and work of the
dairy and farm, but also with the inspiration for their work.

The first annual report of the Agricultural Experiment
Station of the University of Wisconsin, published after the
appointment of the bacteriologist, contained four papers by
him; one on the relation of bacteria to milk and three on
bovine tuberculosis, two lines which were to be followed
actively for many years. In each subsequent report of the
Station and in a number of bulletins were presented the re-
sults of the research work of the department.

It is impossible to review in any detail the things accom-
plished. A few of the chief lines of endeavor will be men-
tioned. In 1892 Robert Koch prepared tuberculin, a product
that was not to fulfill the hopes of him and his friends as a
therapeutic agent, but whose value as a diagnostic agent in
the case of bovine tuberculosis was soon recognized. The
college herd was tested in February, 1894. This test was
one of the first made in this country and the first made west
of the Allegheny Mountains. Twenty-five of the thirty
animals comprising the herd were found to be diseased. The
whole herd was destroyed and a new herd formed which for
twenty years has served as an example to the farmers of the
state of what can be done in the control of this disease. The
destruction of the college herd was a serious step and one
that would have been authorized only by a far-secing adminis-
trator as the history of the Experiment Station and College
of Agriculture has and is showing Professor Henry to have
been. The tendency then as now was to temporize with the
disease. No question remains as to the wisdom of the decision
made in 1894 with reference to the fate of the college herd.
Twenty years later sister institutions were still hesitating
about doing with their herds what they were advising the
farmer to do with his.

The pioneer in any line of human endeavor is not likely
to see the fruition of his work. To no one is this more likely
to happen than to the investigator in agricultural fields. Facts
are discovered, the importance of which is most evident to
those whose background of knowledge enables them to appre-

SCIENTIFIC WORK OF HARRY LUMAN RUSSELL 13

ciate their bearing. Those without such a background must
accept them on faith. The natural and proper conservatism
of the farmer leads him to accept new facts only after their
value has been demonstrated. Yet delay in their acceptance
may often mean great loss and irreparable damage. This
fact is well illustrated by bovine tuberculosis.

The lack of recognition of the importance of bovine tuber-
culosis by the farmer has enabled the disease to gain greater
and greater headway. In 1894 great areas of our country
were free from the disease which now exists in every locality
and is still spreailing, due to the fact that the importance
of the truths pointed out twenty-five years ago is not yet
realized by the great mass of farmers.

If the breeders and the farmers of the state and of the
country had accepted and followed the advice given in the
first papers published on bovine tubereulcsis by the Wisconsin
College of Agriculture, the conditions today would be far
different. It was recognized that the pure bred herds were
the chief centers frcm which the disease was being dissem-
inated. It was recognized that a healthy herd would be a
great asset to its owner. But few of the breeders of the state
saw fit to follow the advice. Those who did have never re-
gretted it.

The knowledge that was obtained concerning the conditions
that existed in the state with reference to bovine tuberculosis
made possible the formulation of a campaign against it.
Briefly, this consisted of an educational propaganda which
should bring to the farmer evidence of the economic im-
portance of the disease, which should acquaint him with the
tuberculin test and its value in keeping tuberculosis out of
his herd or in eliminating it therefrom.

It was recognized that by far the greater part of the dairy
herds of the state were free from the disease, especially in
the more newly settled regions of the state, that the important
and immediate thing was not the elimination of the disease
from the infected herds, but the prevention of its introduction
into healthy herds. In furtherance of the plan of action,
animals that had reacted to the tuberculin test were slaugh-

14 UNIVERSITY OF WISCONSIN STUDIES

tered at various gatherings of farmers and thus there was
presented to them evidence of the ravages which the disease
may occasion in an apparently healthy animal.

If progress was to be made, the tuberculin test must be
widely used. The limited number of veterinarians available
for the work made evident the necessity of extending its appli-
cation to the laity. With this idea in mind, special instruc-
tion in the use of the test was given to the students of the
agricultural college, both in the regular and in the short
course.

It was suggested that the prevention of the sale of tuber-
cular animals would be the most effective way of stopping
the spread of the disease. The passage of a law was urged
that would require the testing of all cattle sold for breeding
and dairy purposes. Such a law was enacted.

The first of these steps was opposed by the veterinarians
who felt that it was an encroachment on their field; the
second was opposed by the farmers who had not been or who
would not be convinced of the importance of the disease. The
plan was too early to meet with the approval of those most
concerned, and by the time they had become ready to give to
such a plan their sympathy and approval, it was too late for
such or any plan to have much effect.

Looking backward from the vantage point of present knowl-
edge, one can see how great a factor the plan would have
been in preventing the spread of the disease which is rapidly
becoming the most important as far as the economic and sani-
tary aspects of the dairy are concerned.

Dean Russell served as a member of the State Live Stock
Sanitary Board for a number of years. The work in bovine
tuberculosis led to an interest in human tuberculosis. The
services rendered in this field were in connection with the
establishment of the State Tuberculosis Sanatorium at Wales.
Dean Russell served as president of the advisory board of
this institution.

For a number of years, work was done for the State Board
of Health in the sanitary examination of water and in the
diagnosis of tuberculosis, diphtheria, and typhoid. In 1903

SCIENTIFIC WORK OF HARRY LUMAN RUSSELL 15

the State Hygienic Laboratory was created by the legislature.
Dean Russell was made director, a position he held until 1908.
Another field in which pioneer work was done was in the
pasteurization of milk and cream for direct consumption.
Bulletin 44 of the Experiment Station was issued in 1895.
The method suggested therein conforms with one exception
to that which is accepted by the health officials as the best
today. The milk was treated in an apparatus that insured
the heating of all of the milk to the desired temperature and
for the desired time. This is nothing more or less than the
‘‘holding’’ method of pasteurization so widely used today.
The temperature recommended was 155° F. for 20 minutes.
At this time the tubercle bacillus was considered to be much
more resistant to heat than was later found to be the case.
For many years pasteurization of milk for direct consump-
tion did not meet with the approval of health officials. They
considered it in the class with preservatives. The process
was gradually introduced into the milk trade by the dis-
tributors whose interest lay in imparting to their product a
better keeping quality. They adopted the machines that had
been found satisfactory in the pasteurization of cream for
butter making. These machines subjected the milk to a high
temperature for a short period, the ‘‘flash’’ method of pasteur-
ization. This process did not insure the freedom of the milk
from pathogenic organisms since a portion was subjected to
the maximum temperature for such a short period that the
organisms therein might not be destroyed. The recognition
of this fact by health officials did not lead them to favor
pasteurization of milk for direct consumption. Indeed, for
many years, the whole weight of influence of the medical pro-
fession was against the pasteurization of milk. They failed
to recognize the trend of affairs in the provisioning of our
cities with milk, that it was becoming increasingly difficult to
obtain a sufficient supply of milk close to the point of con-
sumption, that as supplies must be sought farther away, the
period between the moment of production and of consumption
lengthened. It became increasingly difficult to retard the
process of souring of the milk by the methods then used

16 UNIVERSITY OF WISCONSIN STUDIES

without increasing the price of the product to an extent
that would limit its use. It became more and more evident
that freedom of the municipal supplies of milk from
pathogenic organisms could never be insured by inspection
at the point of production. These two facts indicated to
every opened-minded person the necessity of pasteurization
of milk. The medical profession is on the one hand the most
radical group and on the other the most conservative. As a
group the medical men could see no use, no advantage, in the
pasteurization of milk and until the process was favored by
the physicians, its extension was slow.

The work done from 1894 to 1900 laid the foundation for
the rational pasteurization of milk and cream. It involved
a study of the effect of heat on the physical properties of
milk and a study of the resistance of the tuberele organism
to heat. It was shown that milk could be so treated as to
insure its freedom from pathogenic organisms and yet retain
its original chemical and physical properties to such an extent
as not to mike it undesirable from the standpoint of the
distributor or the consumer. The development of the pasteur-
izine process has been along the lines that were laid down
in the first publication on this subject from the Wisconsin
Experiment Station. Many of the facts discovered and the
points emphasized did not exert the influence they deserved
to exert. They were rediscovered by later workers at a time
when the world was ready to aceept them. This is the fate
of the pioneer in scicnee.

At the time Dean Russell came to the Experiment Station,
research work on the factors concerned in the production of
cheddar cheese was being carried on under the leadership
of Dr. 8. M. Babeock. The first bacteriological paper in this
field, Gas-Producing Bacteria and the Relation of the Same to
Cheese, was published in the report of the Experiment Station
for 1895. This was quickly followed by other papers dealing
with the bacterial flora of cheese and their réle in the ripen-
ine of cheese and the importance of the quality of milk in the
cheese industry. The development of the Wisconsin Curd Test
gave to the cheese maker a means of testing the milk delivered

SCIENTIFIC WORK OF HARRY LUMAWN RUSSELL 17

by the patrons for harmful types of bacteria. This method
furnished the cheese maker a reasonable way of overcoming
the most frequently occurring and most important trouble
that confronted him.

The field of enzymology was a relatively unexplored one
in 1897, when a report was made by Dr. Babcock and Dean
Russell on the presence in milk of a proteolytic enzyme to
which the term galactase was applied. A study of the oc-
currence of this enzyme showed it to be present in the milk
of various species of animals. The study of the properties
of this enzyme and its réle in the ripening of cheddar cheese
led to a modification in the curing of cheese that has become
of the greatest importance to the cheese industry. The
ripening of cheese at low temperatures was held by men of
long practical experience to be impossible. The results of the
experiments made at Wisconsin showed that at much lower
temperatures than had been used in the curing rooms, the
occurrence of abnormal flavors was much less frequent than
at the higher temperatures. The experiments in the cold
curing of cheese were extended in 1901 and 1902 and later
were carried on in cooperation with the Dairy Division of
the U. S. Department of Agriculture and the New York Ex-
periment Station at Geneva. This work has served to revolu-
tionize the entire business of curing cheese. It has saved and
is saving every year large sums of money to the industry
through the prevention of abnormal cheese. It is an ex-
cellent example of the practical results that may follow from
work that apparently can promise no practical results.

In the early years of this century the presence of cellular
elements in milk was attracting the attention of health officials.
Rules were being formulated that meant the rejection of a
large part of the milk produced. The rules had been for-
mulated out of a clear sky. Their authors thought that milk
should contain not to exceed a certain arbitrary number of
cells. They had not taken the trouble to determine by a study
of milks anything concerning the normal content in these
elements and some of the factors that influence their number.
Work done on the University herd showed that the standards

2

18 UNIVERSITY OF WISCONSIN STUDIES

that had been proposed were far too exacting, that what were
being considered abnormal conditions in milk were really
normal. The modern views of the significance of the cellular
elements in milk were forecast in the papers published in
1906 and 1907.

Another field in which pioneer work was done was that
of the relation of bacteria to diseases of plants. The views
held by those highest in the field of plant pathology were that
bacteria could not be of importance in the causation of disease
in plants due to the acidity of the plant juice. It was known
that bacteria as a group grow best in nutrient solutions that
are alkaline to litmus. Plant juices are usually acid to litmus,
therefore the inference was drawn that bacteria could not
grow in the plant tissues. Again the structure of the plant
did not seem to favor the entrance of bacteria or their spread
in the tissues of the plant. The German investigators of
1895 in bacteriology were not accustomed to pay much atten-
tion to what had been done in America. One of the first
bacteriological investigations in this country was that of Dr.
T. J. Burrell of the University of Illinois on pear blight.
This should at least have indicated to the bacteriologists the
possibility of bacterial diseases of plants.

The training which Dean Russell had received in botany
and in connection with the preparation of his doctor’s thesis
had given him an admirable preparation to undertake an
investigation of a disease that was causing great havoc in
the cabbage-growing sections of Wisconsin. In 1898 Bulletin
65 of the Experiment Station was published. It was entitled
A Bacterial Rot of Cabbage and Allied Plants. This work
is to be classed as one of the first extensive pieces of research
in a field that has assumed so great an importance.

The period from 1893 to 1907 was crowded with work of
instruction and research; the period from 1907 to 1917, with
executive duties whose manifold demands have made im-
possible any active continuance in the former lines of work.
This condition is to be regretted, on the one hand, for it has
robbed the students of the college of an inspiring instructor,
ef whom there are few, and the world of an able research man,

SCIENTIFIC WORK OF HARRY LUMAN RUSSELL 19

but it has given to the state a successful administrator of its
College of Agriculture and its Experiment Station. It is
fortunate for the state that energies have not been dissipated
by attempting to follow many lines, but that they have been
confined to one line, that of administration.

As earlier stated, the University of Wisconsin has been
fortunate in the loyalty of its staff. This has been noteworthy
in every division of the University. For thirty-four years
the destiny of the agricultural work of the University has
been in the hands of but two men. It is to be hoped that the
same may be said when the half-century is reached.

BOOKS AND SCIENTIFIC PAPERS OF H. L. RUSSELL

Books

Outlines of Dairy Bacteriology, 1894. This has appeared in
ten different editions, the last two in cooperation with
Professor E. G. Hastings.

Public Water Supplies, Turneaure and Russell, 1901.

Experimental Dairy Bacteriology, Russell and Hastings, 1909.

Agricultural Bacteriology, Russell and Hastings, 1915.

PAPERS

Preliminary Observations on the Bacteria of Ice from Lake
Mendota, Madison, Wisconsin in Mcdical News, p. 15,
1889.

Observations on the Temperature of Trees in Bot. Gaz. 14,
pp. 216-222, 1889.

Penicillium and Corrosive Sublimate in Bot. Gaz. 15, pp. 211-
212, 1890.

Bactcrial Investigations of the Sea and Its Floor in Bot. Gaz.
17, pp. 312-321, 1892.

Impungversuche mit Giard’s pathogenem Leuchtbacillus in
Centbl. Bakt. (ete.), I. Abt., 11, pp. 557-559, 1892.
The Effect of Mechanical Moveimcnt on the Growth of Cer-

tain Lower Organisms in Bot. Gaz. 17, pp. 8-15, 1892.

Untersuchungen tiber im Golf von Neapel lebende Bakterien
in Zettschr. f. Hyg., 11, pp. 165-206, 1892.

Bacteria in Their Relation to Vegetable Tissue. in Johns Top-
kins Hospital Reports, 3, pp. 223-263, 1893.

Non-parasitic Bacteria in Vegetable Tissue in Bot. Gaz. 18.
pp. 938-96, 1893.

The Bacterial Flora of the Atlantic Ocean in the Vicinity of
Wood’s Holl, Mass. in Bot. Gaz. 18, pp. 383-395, 411417,
439-447, 1893.

BOOKS AND SCIENTIFIC PAPERS BY H. L. RUSSELL 21

On the Efficiency of Tuberculin as a Diagnostic Agent in
Tuberculosis in 11th Ann. Rept., Wis. Agr. Exp. Sta.,
pp. 166-197, 1894.

The Firation of Free Nitrogen by Plauts in Bot. Gaz. 19,
284-293, 1894.

The Infectiousness of Milk from Tuberculous Cows in 11th
Ann. Rept., Wis. Agr. Exp. Sta., pp. 196-200, 1894.
The Relation of Separator Sime to Tuberculosis in Hogs in

ith Ann, Rept., Wis. Agr. Exp. Sta., pp. 201-204, 1894.

The Source of Bacterial Infection of Milk and the Relation
of the Same to the Keeping Qualities of Milk in 11th
Ann. Rept., Wis. Agr. Exp. Sta., pp. 150-165, 1894.

Tuberculosis and the Tuberculin Test, Bul. 40, Wis. Agr.
Exp. Sta., 47 p., 1894.

Tuberculosis at the Wisconsin Experiment Station in Vet.
Magazine, Aug. 1894. (With W. G. Clark.)

-l Biological Study of Pasteurized Milk and Cream under
Commercial Conditions in Centbl. Bakt. (ete.), II. Abt.,
1, pp. 741-751, 1895. ‘

Gaseous Ferments in the Canmng Industry in 12th Ann.
Rept., Wis. Agr. Exp. Sta., pp. 227-231, 1895.

Gas Producing Bacteria and the Relation of the Same to
Cheese in 12th Ann. Rept., Wis. Agr. Exp. Sta., pp. 139-
150, 1895.

Investigations on Bacterva in Bot. Gaz. 20, pp. 419-422, 1895.

Notes on the Pasteurization of Milk in 12th Ann. Rept., Wis.
Agr. Exp. Sta.. pp. 158-173, 1895.

Pasteurization of Milk and Cream for Direct Consumption,
Bul. 44, Wis. Agr. Exp. Sta., 48 p., 1895.

The Influence of Acid on the Texture of Cheese in 12th Ann.
Rept., Wis. Agr. Exp. Sta., pp. 129-133, 1895. (With J.
W. Decker.)

The Influence of Aération on the Flavor of Tainted Curds in
Cheese Making in 12th Ann. Rept., Wis. Agr. Exp. Sta.,
pp. 127-129, 1895.

The Use of Bacterial Cultures in Butter Making with Espec-
ial Reference to the Conn Culture (B. 41) in 12th Ann.
Rept., Wis. Agr. Exp. Sta., pp. 174-226, 1895. (With E.
H. Farrington.)

22 UNIVERSITY OF WISCONSIN STUDIES

Typhoid Fever Disseminated through the Milk Supply in
Science, pp. 682-683, Nov. 15, 1895.

Conditions Affecting the Consistency of Milk in 13th Ann.
Rept., Wis. Agr. Exp. Sta., pp. 73-80, 1896. (With 8. M.
Babcock.)

On the Restoration of the Consistency of Pasteurized Milk
and Cream in 13th Ann, Rept., Wis. Agr. Exp. Sta., pp.
81-94, 1896. (With S. M. Babcock.)

Pure Lactic Cultures in Cheese Making in 13th Ann. Rept.,
Wis. Agr. Exp. Sta., pp. 112-126, 1896.

The Conn Culture (B. 41) in Butter Making, Bul. 48, Wis.
Agr, Exp. Sta., 22 p., 1896. (With E. H. Farrington.)

Lhe Pasteurization of Milk in Pharmaccutical Review 14,
May, 1896.

The Restoration of the Consistency of Pasteurized Cream,
Bul. 54, Wis. Agr. Exp. Sta., 8 p., 1896. (With S. M.
Babcock.)

The Restriction of Tuberculosis by Isolation and the Use of
Affected Animals for Breeding Purposes in 13th Ann.
Rept., Wis. Agr. Exp. Sta., pp. 127-133, 1896.

The Rise and Fall of Bacteria in Cheddar Cheese in 13th Ann.
Rept., Wis. Agr. Exp. Sta., pp. 95-111, 1896.

Tubcreulin Inoculations for the Year 7896 in 18th Ann.
Rept., Wis. Agr. Exp. Sta., pp. 184-137, 1896.

Influence of Temperature on the Ripening of Cheese in 14th
Ann, Rept., Wis. Agr. Exp. Sta. pp. 194-210, 1897.
(With 8. M. Babcock.)

Note on the Action of Chemicals on Organisms in Bot. Gaz.
23, pp. 223-224, 1897.

Tainted or Defective Milks: their Causes and Methods of
Prevention, Bul. 62, Wis. Agr. Exp. Sta., 27 p., 1897.

Lhe Checse Industry: Its Development and Possibilities in
Wisconsin, Bul. 60, Wis. Agr. Exp. Sta., 24 p., 1897.
(With S. M. Babcock.)

Lhe Distribution of Cheese Factories in Wisconsin in 1 ith
Ann. Rept., Wis. Agr. Exp. Sta., pp. 149-152, 1897.
(With S. M. Babcock and J. W. Decker.)

BOOKS AND SCIENTIFIC PAPERS BY H. L. RUSSELL 23

Lhe Rise and Fall of Bacteria in Cheddar Cheese in Centbl.
Bakt. (ete.), II. Abt., 3, pp. 456-467, 1897. (With John
Weinzirl. )

Tolerance of Certain Milk Bacteria toward Ether in
14th Ann. Rept., Wis. Agr. Exp. Sta., pp. 211-215, 1897.
(With 8S. M. Babcock.)

Unorganized Ferments of Milk; a new Factor in the Ripen-
ing of Cheese in 14th Ann. Rept., Wis. Agr. Exp. Sta.,
pp. 161-193, 1897. (With 8S. M. Babcock.)

Unorganized Ferments of Milk; a new Factor in the Ripen-
ing of Cheese in Centbl. Bakt. (ete.), II. Abt., 3, pp. 615—
620, 1897. (With S. M. Babcock.)

A Bacterial Rot of Cabbage and Allied Plants, Bul. 65, Wis.
Agr. Exp. Sta., 39 p., 1898.

An Improved Curd Test for the Detection of Tainted Milk in
15th Ann. Rept., Wis. Agr. Exp. Sta., pp. 45-53, 1898.
(With S. M. Babcock and J. W. Decker.)

Distribution of Galactase in Cow’s Milk in 15th Ann. Rept.,
Wis. Agr. Exp. Sta. pp. 87-92, 1898. (With 8S. M.
Babcock and Alfred Vivian.)

Distribution of Galactase in Different Species of Mammalia
in 15th Ann Rept., Wis Agr. Exp. Sta., pp. 938-97, 1898.
(With S. M. Babcock and Alfred Vivian.)

Factory Tests for Milk, Bul. 67, Wis. Agr. Exp. Sta., 20 p.,
1898. (With 8. M. Babcock and J. W. Decker.)

Pasteurization as Applied to Butter Making, Bul. 69, Wis.
Agr. Exp. Sta., 40 p., 1898. (With E. H. Farrington.)

Pasteurization Experiments in Butter Making in 15th Ann.
Rept., Wis. Agr. Exp. Sta., pp. 54-70, 1898. (With E.
H. Farrington. )

Propagation of Starter for Ripening Cream in 15th Ann
Rept., Wis. Agr. Exp. Sta., pp. 73-76, 1898.

Properties of Galactase, a Digestive Ferment of Milk in 15th
Ann. Rept., Wis. Agr. Exp. Sta., pp. 77-86, 1898. (With
S. M. Babcock and Alfred Vivian.)

Relative Absorption of Odors in Warm and Cold Milk in 15th
Ann. Rept., Wis. Agr. Exp. Sta., pp. 104-109, 1898.
Sticky and Slimy Bread and Its Causes in 15th Ann. Rept.,

Wis. Agr. Exp. Sta., pp. 110-113, 1898.

24 UNIVERSITY OF WISCONSIN STUDIES

The Antiseptic Value of Certain Chemicals in Milk in 15th
Ann. Rept., Wis. Agr. Exp. Sta, pp. 98-103, 1898.
(With S. M. Babcock and Alfred Vivian.)

Effect of Different Influences on the Normal Temperature of
Cattle in 16th Ann. Rept., Wis. Agr. Exp. Sta., pp. 194-
204, 1899. (With V. H. Bassett.)

Effect of Digesting Bacteria on the Cheese Solids of Milk in
16th Ann. Rept., Wis. Avr. Exp. Sta., pp. 187-193, 1899.
(With V. H. Bassett.)

Influence of Galactase in the Ripening of Cottage Cheese in
16th Ann. Rept., Wis. Agr. Exp. Sta., pp. 175-178, 1899.
(With 8. M. Babcock and Alfred Vivian.)

Pasteurization of Milk and Cream at 140° F. in 16th Ann.
Rept., Wis. Agr. Exp. Sta., pp. 129-1389, 1899. (With E.
H. Farrington.)

The Action of Proteolytic Ferments on Milk with Special
Reference to Galactase, the Cheese-Ripening Enzyme in
16th Aun. Rept., Wis. Agr. Exp. Sta., pp. 157-174, 1899.
(With S. M. Babeock, Alfred Vivian, and E. G. Hast-
ings. )

The History of a Tuberculous Herd of Cows, Bul. 78, Wis.
Agr. Exp. Sta., 16 p., 1899.

The Significance of Certain Gas-producing Bacteria of Non-
Colon Type in Sanitary Water Analysis in Trans. Am.
Pub. Health Asso., 25,1900. (With V. H. Bassett.)

A Closed Circuit Respiration Apparatus in 17th Ann. Rept.,
Wis. Agr. Exp. Sta., pp. 142-146, 1900. (With S. M.
Babcock.)

Causes Operative in the Formation of Silage in 17th Ann.
Rept., Wis. Agr. Exp. Sta., pp. 123-141, 1900. (With 8S.
M. Babcock.)

Galaktasc, das der Milch eigentiimliche proteolytische Fer-
ment, seine Kigenschaften wnd seine Wirkung auf dic
Proteide der Milch in Centbl. Bakt. (ete.), II. Abt., 6,
pp. 17-21, 45-50, 79-88, 1900. (With S. M. Babcock. )

Influence of Rennet on Cheese Ripening in 17th Ann. Rept.,
Wis. Agr. Exp. Sta., pp. 102-122, 1900. (With s. M.
Babeock and Alfred Vivian.)

BOOKS AND SCIENTIFIC PAPERS BY H. L. RUSSELL 20

Outbreak of Anthrax Fever Traceable to Tannery Refuse in
17th Ann, Rept., Wis. Agr. Exp. Sta., pp. 171-184, 1900.

Relation of the Enzymes of Rennet to Ripening of Cheddar
Cheese in Centbl. Bakt. (ete.), II. Abt., 6, pp. 817-825,
1900. (With 8. M. Babcock.)

Thermal Death Point of Tubercle Bacilli under Commercial
Conditions in 17th Aun. Rept., Wis. Agr. Exp. Sta., pp.
147-170, 1900. (With E. G. Hastings.)

Bovine Tuberculosis and Milk Supplies in The Philadelphia
Med. Journal, Nov. 1901.

Bovine Tuberculosis in Wisconsin, Bul. 84, Wis. Agr. Exp.
Sta., 16 p., 1901. (With E. G. Hastings.)

Causes Operative in the Formation of Silage in 18th Ann.
Rept., Wis. Agr. Exp. Sta., pp. 177-184, 1901. (With
8S. M. Babcock. )

Influence of Sugar on the Nature of the Fermentation Oc-
curring in Milk and Cheese in 18th Ann. Rept., Wis.
Agr. Exp. Sta., pp. 162-176, 1901. (With S. M. Babcock,
A. Vivian, and E. G. Hastings.)

Influencing of Cold-Curing on the Quality of Cheese in 18th
Ann. Rept., Wis. Agr. Exp. Sta., pp. 136-162, 1901.
(With S. M. Babeock, A. Vivian, and U. 8. Baer.)

On the Increased Resistance of Bacteria in Milk Pasteurized
im Contact with the Air in 18th Ann. Rept., Wis. Agr.
Exp. Sta., pp. 185-194, 1901. (With E. G. Hastings.)

The Nature and Cause of Communicable Diseases, Bul. 7,
Wis. State Dept. of Education, 63 p., 1901. (With W.
D. Frost.)

A Micrococcus, the Thermal Death Iimit of which is 76° C.
in Centbl. Bakt. (ete.) II. Abt., 8, pp. 339-342, 1902.
(With E. G. Hastings.)

Conditions Affecting the Development of White Specks in
Cold-Cured Cheese in 19th Ann. Rept., Wis. Agr. Exp.
Sta., pp. 180-183, 1902. (With S. M. Babcock, A.
Vivian, and U. S. Baer.)

Curing of Cheddar Cheese with Especial Reference to Cold-
Curing, Bul. 94, Wis. Agr. Exp. Sta., 44 p., 1902. (With
S. M. Babcock.)

26 UNIVERSITY OF WISCONSIN STUDIES

Die bei der Herstellung von Gérfutter (Silage) wirkenden
Ursachen in Centbl. Bakt. (etc.), II. Abt., 9, pp- 81-88,
1902. (With S. M. Babcock.)

Einfluss des Zuckers auf die Nature der in der Mulch und
dem Kise vor sich gehenden Gérung in Centbl. Bakt.
(ete.), II. Abt., 9, pp. 757-768, 1902. (With S. M.
Babcock. )

History of the Epidemic of Typhoid Fever at Baraboo, Wis.
in 19th Bien. Rept. Wis. State Bd. of Health, pp. 117-
130, 1902.

Influence of Cold-Curing on the Quality of Cheddar Cheese
(2nd paper) in 19th Ann. Rept., Wis. Agr. Exp. Sta.,
pp. 150-164, 1902. (With S. M. Babcock, A. Vivian,
and U.S. Baer.)

Influence of Temperatures Approximating 60° F. on the
Development of Flavor in Cold-Cured Cheese in 19th.
Ann. Rept., Wis. Agr. Exp. Sta., pp. 165-173, 1902.
(With S. M. Babcock, A. Vivian, and U. S. Baer.)

Influence of Varying Quantities of Rennet on Cold-Cured
Cheese in 19th Ann. Rept., Wis. Agr. Exp. Sta., pp. 174-
179, 1902. (With S. M. Babcock, A. Vivian, and U. S.
Baer.)

On the Increased Resistance of Bacteria in Milk Pasteurized
in Contact with the Air in Centbl. Bakt. (ete.), II. Abt.,
8, pp. 462-469, 1902. (With E. G. Hastings.)

Influence of the ‘‘Scalded Layer’’ on the Efficiency of Pas-
teurization of Milk in Rev. Gen. Latt., 3, pp. 84-39, 49-
56, 1908. (With E. G. Hastings.)

Shrinkage of Cold-Cured Cheese during Ripening in Bul. 101,
Wis. Agr. Exp. Sta., 30 p., 1903. (With S. M. Babcock
and U. 8. Baer.)

A Lesson in Bovine Tuberculosis, Bul. 114, Wis. Agr. Exp.
Sta., 8 p., 1904.

Effect of Short Periods of Exposure to Heat on Tubercle
Bacilli in Milk in 21st Ann. Rept., Wis. Agr. Exp. Sta.,
pp. 178-192, 1904. (With E. G. Hastings.)

Disappearance of Bacteria Artificially Introduced into Cows’
Udders in 21st Ann. Rept., Wis. Agr, Exp. Sta.,
pp. 164-168, 1904. (With E. G. Hastings.)

BOOKS AND SCIENTIFIC PAPERS BY H. L. RUSSELL 27

Infectiousness of Milk from Tubercular Cows in 21st Ann.
Rept., Wis. Agr. Exp. Sta., pp. 172-177, 1904. (With E.
G. Hastings.)

Relation of Flavor Development in Cold-Cured Cheddar
Cheese to Bacterial Life in the Same in 21st Ann. Rept.,
Wis. Agr. Exp. Sta., pp. 155-163, 1904. (With E. G.
Hastings.)

The Cold-Curing of Cheese, Bul. 49, U. 8. Dept. Agr., 70 p.,
1903. (With 8. M. Babcock and U. S. Baer.)

The Influence of Sugar on Fermentations in Milk and Milk
Curds in Archiv. Pediatrics, Oct., 1904.

The Longevity of the Typhoid Bacillus in Water in Jour.,
Infect. Diseases 1, pp. 641-689, 1904. (With E. O. Jor-
dan and F. R. Zeit.)

A Swiss Cheese Trouble Caused by a Gas-Forming Yeast,
Bul. 128, Wis. Agr. Exp. Sta., 26 p., 1905. (With E. G.
Hastings.)

Bacteriological Test of a Bottle-Washing Device in 22nd Ann.
Rept., Wis. Agr. Exp. Sta., pp. 227-231, 1905. (With C.
Hoffmann.)

Inoculation Experiments with Alfalfa and Soy Beans in
2and Ann. Rept., Wis. Agr. Exp. Sta., pp. 242-261,
1905. (With R. A. Moore.)

On the Detection of a Tainted Condition in Pasteurized Milk
in 22nd Ann. Rept., Wis. Agr. Exp. Sta., pp. 222-226,
1905. (With C. Hoffmann.)

Report of the Wisconsin Tuberculosis Commission, 1905.

Studies on the Pasteurization of Milk in a ‘‘Continuous+
flow’? Machine in 22nd Ann. Rept., Wis. Agr. Exp.
Sta., pp. 232-241, 1905. (With C. Hoffmann.)

Two Ways of Treating Tuberculosis in a Herd, Bul. 126,
Wis. Agr. Exp. Sta., 15 p., 1905.

Development of Factory Dairying in Wisconsin in 23d Ann.
Rept., Wis. Agr. Exp. Sta., pp. 100-106, 1906. (With
U. S. Baer.)

Distribution of Tuberculosis in Suspected and Non-Suspected
Herds in Wisconsin, Bul. 133, Wis. Agr. Exp. Sta.,
14 p., 1906. (With E. G. Hastings.)

28. UNIVERSITY OF WISCONSIN STUDIES

Development of Factory Dairying in Wisconsin, Bul. 140,
Wis. Agr. Exp. Sta., 19 p., 1906. (With U. S. Baer.)
Tuberculosis Work for 1905-1906 in 23d Ann. Rept., Wis.

Agr. Exp. Sta., pp. 91-99, 1906. (With E. G. Hastings.)

Director’s Report for 1907 in 24th Ann. Rept., Wis. Agr.
Exp. Sta., pp. 1-24, 1907.

Distribution of Cell Elements in Milk and their Relation to
Sanitary Standards in 24th Ann. Rept., Wis. Agr. Exp.
Sta., pp. 231-253, 1907. (With C. Hoffmann.)

Leucocyte Standards and the Leucocyte Content of Milks
from Apparently Healthy Cows in Jour. Infect. Diseases,
Supplement No. 8, pp. 63-75, 1907. (With C. Hoff-
mann.)

Tuberculosis Tests for 1906-1907 in 24th Ann. Rept., Wis.
Aer. Exp. Sta., pp. 224-230, 1907.

The Spread of Tuberculosis through Factory Skim Mik, Bul.
143, Wis. Agr. Exp. Sta., 28 p., 1907.

Effect of Heating wpon the Determination of Leucocytes in
Mitk in Am. Jour. Pub. Hyg. 18, pp. 285-291, 1908.
(With C, Hoffmann.)

Tuberculosis of Domestic Stock and Its Control, Special Bul.,
Wis. Agr. Exp. Sta., 8 p., 1908.

Vaccination against Tuberculosis in Cattle, Bul. 165, Wis.
Agr. Exp. Sta., 13 p., 1908. (With C. Hoffmann.)

A Catechism on Bovine Tuberculosis, Cir. 28, Wis. Agr. Exp.
Sta., 24 p., 1911. (With E. G. Hastings.)

The “Coming of Age’’ of the Babcock Test, Cir. of Inforin.
32, Wis. Agr. Exp. Sta., 18 p., 1912.

Director’s Reports 1908-1916; Wis. Agr. Exp. Bulletins
171, 193, 203, 218, 228, 240, 250, 268, 275.

THE DEVELOPMENT OF CITY MILK SUPPLY ~
PROBLEMS

H. A. Harpine

While records of fermentative changes in milk and its
products extend back practically to the dawn of history, little
significance can be attached to observations of the details of
germ life in the dairy prior to the beginning of modern bacter-
iological technique in 1881.1

About 1890 there was a widespread development of in-
terest regarding germ life in the dairy. Adametz? in Austria
and de Freudenreich? in Switzerland were then at work on
cheese problems, Storch* in Denmark and Weigmann® in
Germany were busy with the relations between germ life and
flavors in butter, while in America Conn* was examining the
bacteria in cream and Sedgwick’ had made some observations
on the bacteria in city milk.

In connection with the World’s Fair in Chicago in 1893,
Conn displayed a collection of dairy bacteria illustrating the
changes which these germs were able to produce in milk and
cream. Whatever else this exhibit may have accomplished, it
was an important factor in convincing Dean Henry that

1R. Koch, Zur Untersuchung von pathogenen Organismen in Mitt, aus
dem Kaiserl. Gesundheitsamte, 1, pp. 1-18, 1881.

2L, Adametz, Bakteriologische Untersuchungen iiber den Reifungspro-
zess der Kiise in Land. Jahr., 18, pp. 227-270, 1889.

2Hd. v. Freudenreich, Bakteriologische Untersuchungen tiber den Reif-
angsprozess des Emmenthalerkdses in Milch, Zeit., 21, pp. 368-369, 1892.

4vV. Storch, 18 Beretning kgl. Veter. og Landbohéjskoles Laborat. f.
landdkon. Forség. Kjobenhavn., 1890.

5H. Weigmann, Landw. Wochenbl. f. Schleswig-Holstein, 40, p. 549,
1890. Also Milch Zeit., 19, p. 598, 1890.

6H. W. Conn, Bacteria in Milk, Cream and Butter in Storrs Agr. Exp.
Station Ann. Rept., 2 (1889), 52-67, 1890.

™W. T, Sedgwick and Batchelder, A Bacteriological Examination of the
Boston Milk Supply in Boston Medical and Surgical Journal, 126, pp.
25 ff., 1892.

30 UNIVERSITY OF WISCONSIN STUDIES

here was a field of study which should be further cultivated
in behalf of the dairy industry. The result was the appoint-
ment of Dr. H. L. Russell as head of the department of
bacteriology in the University and Bacteriologist to the Agri-
cultural Experiment Station. The uniqueness of this action
may be judged from the fact that at that time there was no
similar official position in any American agricultural experi-
ment station. The nearest analogy was Dr. H. W. Conn,
professor of biology at Wesleyan University, Middleton,
Conn., who was doing some cooperative work with the Storrs
Agricultural Experiment Station and the Federal Depart-
ment of Agriculture.

To those coming recently into the field of dairy bacteriology
it is difficult to picture the meagreness of the facts available
to Dr. Russell when he was inducted into this field of work
in September, 1893. These available facts may be fairly
summarized as follows:

1. Bacteria were known to be widely distributed in nature
and to find their way accidentally into milk.

2. They multiplied in milk and in connection with their
growth produced various compounds among which acid was
the most evident. The work of Storch, Weigmann, and Conn
suggested that the flavors in cream and butter resulted in
part from such growths. The relation of bacteria to cheese
was less evident.

3. It was known that tuberculosis was caused by a definite
germ and a number of other diseases of cows and people were
more or less definitely ascribed to germ causes.

During the sueceeding twenty-five years Dr. Russell, in
person and through his students, has contributed largely to
increasing our knowledge along all lines of dairy bacteriology.
One of the first problems which engaged his attention was
that of bovine tuberculosis® and the influence he exerted has
helped to bring Wisconsin to the front in the struggle against

i 1

®H. L. Russell, On the Efficiency of Tuberculin as a Diagnostic Agent

in Tubcreulosis in Annual Report, Wis. Agr. Exp. Sta., 11 (1894), DD.
166-195, 1895.

DEVELOPMENT OF CITY MILK SUPPLY PROBLEMS 31

this disease; his clear analysis of B41° and its relation to
butter flavors saved the creamery industry large sums of
money; his work in connection with cheese ripening has not
only thrown much light upon the intricacies of this problem
but out of these studies has come the cold curing of cheese*®
which has been of untold advantage to the cheese industry;
and his influence in the field of city milk supplies will be
presented in some detail in this paper.

Because his influence through his own investigations and
those of his students has touched every problem in connection
with city milk supplies during the past twenty-five years this
presentation will be simplified by treating these problems in
the order of their historical development.

City milk supply problems have developed with the growth
of cities. The small city where the necessary milk was pro-
duced within the municipality or upon adjoining farms and
delivered within a few hours was slow to recognize that it had
city milk problems.

The last quarter of the nineteenth century was marked by
a rapid increase in urban population, particularly in the
eastern portion of the United States. Along the coast from
Boston to Washington there developed a succession of rapidly
growing cities with a correspondingly increasing demand for.
milk. The ocean prevented production on one side and the
expanding zone of milk producing territory tributary to each
city soon met, compelling an even more rapid development of
the source of supply to the West and North. As late as 1890
the source of supply for the majority of these coast cities
was essentially local while at the present time practically all
of the accessible territory east of Buffalo and up to and even
across the Canadian border is engaged in supplying them.

The tardy awakening of a consciousness of their milk prob-
lems on the part of the American cities is quite evident from

°m. H. Farrington and H. L. Russell, The Use of Bacterial Culture
Starters, with Especial Reference to the Conn Culture (B41), in Annual
Report, Wis. Agr. Exp. Sta., 12 (1895), pp. 174-226, 1896.

1S, M. Babcock and H. L. Russell, Influence of Temperature on the
Ripening of Cheese in Annual Report, Wis. Agr. Exp. Sta, 14 (1897),
pp. 194-210, 1897.

32 UNIVERSITY OF WISCONSIN STUDIES

another angle. The city can acquire little exact information
regarding the quality of its milk supply and can do even less
in the matter of correcting abuses until it provides a system
of inspection and collection of milk samples. As late as 1890
only about a dozen American cities had made specific pro-
visions for such inspection and examination.”*

In their attempts at handling the milk situation the cities
have given their attention successively to various aspects of
the milk question and for the time being each of these aspects
has heen considered as essentially the milk problem. While
the chronological order in which these subdivisions of the
question have been emphasized in different cities has varied,
the common sequence has been food value, healthfulness,
cleanliness, and keeping quality.

The necessity of determining existing facts with regard to
these problems has quite naturally created the problem of
milk inspection and the complicated results of more recent
milk inspection have emphasized the need of milk grading as
a way of clearly summarizing the situation for the consuming
public.

THE PROBLEM oF Foop VALUE

Since milk is uniformly retailed by volume, there is a
temptation to inerease the volume through the addition of
water. Moreover, the ready market for cream offers an addi-
tional temptation to partially skim the milk before sale. Many
of the early examinations of milk supplies showed that both
practices were fairly common. The earliest recognized
problem of city milk supplies was safeguarding the composi-
tion or food value of the milk.

Massachusetts passed an aet forbidding the watering and
skinmming of milk in 1856 as did New York City in 1873.”
However, little could be accomplished in the absence of simple
and accurate means for detecting these practices. The
lactometer was of scme service in this connection but it was

4H, N. Parker, City Milk Supply, p. 371.
% Tbid., p. 370.

DEVELOPMENT OF CITY MILK SUPPLY PROBLEMS a5

not until after the discovery of the Babcock test in 1890"
that municipal control of skimming and watering became
reasonably efficient. In the decade following 1890 state and
municipal regulations against skimming and watering became
practically universal. As an aid in securing court convictions
minimum legal standards were commonly included in these
regulations. While these minimum legal standards have un-
doubtedly served a useful purpose in combating skimming
and watering they have exerted an unexpected and unfor-
tunate influence because they have tended to standardize the
entire supply at a low level.

The growing appreciation of the wide variation in the
food value of the milk now upon the general market is giving
the problem of food value a new form. There is a desire on
the part of the public for a milk considerably richer in fat
than required by the minimum legal standards. Milk dis-
tributors desire to cater to this trade and wish to distribute
a milk with a uniform fat content distinctly above that of the
ordinary supply. The fat content of the normal milk supply
is subject to variations. A supply which on the average gives
the desired fat content will fall below this desired composi-
tion during a portion of the year and be above the desired
point during another portion. This difficulty could be readily
met by adding cream during one period and skim milk at
another were it not that such additions are a violation of
some of the laws regarding milk adulteration. The proper
regulation of milk standardization is now receiving careful
consideration, and standardization seems destined to be an
early development in the retail milk business.

Until somewhat recently public consideration of food value
of milk concerned itself almost exclusively with the amount
of fat present. Later some attention began to be given to
the accompanying solids, and the total energy value or the
calorific value of the milk came under discussion. The
newest angle of this old question of the food value of milk

2S. M. Babcock, A New Method for the Estinvation of Fat in Milk,
Especially Adapted to Creameries and Cheese Factories in Annual Re-
port, Wis. Agr. Exp. Sta., 7 (1890), pp. 98-113, 1890.

3

34 UNIVERSITY OF WISCONSIN STUDIES

is the recognition of the fact that milk is the most available
source of certain substances indispensable to the growth of
the young and the well being of the adult. Much of the
study of these important, compounds designated as ‘‘fat
soluble a’’ and ‘‘water soluble b’’ has been conducted at Wis-
consin.’* The nutritive importance of these compounds is
emphasized by the fact that recent studies of pellagra make
it evident that milk is practically a specific for this disease.
Pellagra in this country in a single year is variously estimated
at between 150,000 and 200,000 cases, with a mortality of ap-
proximately 5 per cent. These figures suggest that there is
more disease and death in this country every year from the
failure to use sufficient milk than results from the use of bad
milk. While this fact should not militate against all reason-
able efforts to improve the quality of the present milk supply,
it should lead to vigorous efforts to increase the consumption
of fluid milk.

Tur PROBLEM OF HEALTHFULNESS

Healthfulness as here applied to milk refers to the absence
of germs capable of transmitting specific diseases.

The early objections to watering and skimming of milk
were based in part upon the supposition that such practices
rendered the milk unhealthful. When carried to such lengths
as to reduce the amount of food received by the child below
a maintenance ration this conception is in a sense correct.
However, in modern thinking these practices are objected to
rather as a fraud in that they reduce the food value which
the purchaser receives.

Tuberculin as a means of diagnosing bovine tuberculosis
was just beginning to be tried in 1893. A test of the Wisconsin
Experiment Station herd showed it to be largely tuberculous.
Dr. Russell immediately took an important part in the strug-

“ELV. McCollum, The Newer Knowledge of Nutrition, MacMillan, 1918.
% J, Goldberger, G. A. Wheeler and E. Sydenstricker, A Study of the
Dict of Nonpelagrous and of Pelagrous Households in Textile Mill Com-
He Siac in, South Carolina in Jour, Amer. Med. Asso., 71, pp. 944-949,

DEVELOPMENT OF CITY MILK SUPPLY PROBLEMS 35

gle with this disease, a struggle which has continued unabated
for twenty-five years and the end is not yet. He promptly
concluded that the tuberculin test properly conducted was
the best available means of diagnosis.**° He was among the
first, if not the first, in America to recognize the value of the
Bang Method in treating valuable herds.1*

He was quick to recognize the need of some method of
rendering the public milk supply safe from the danger of
carrying tuberculosis to the consumers. He turned at once
to pasteurization’® as a safeguard and in the succeeding years
no more practical method of accomplishing this purpose
has been discovered. Pasteurization then labored under the
disadvantage that the available data on the thermal death
point of the tubercle bacillus called for a time and temperature
of pasteurization which seriously impaired the commercial
qualities of the milk. As a result pasteurization as it was
done commercially up to 1900 was rarely satisfactory.

In 1898 Theobald Smith’? published his studies on the
thermal death point of the tubercle bacillus showing that in
laboratory tests the germ of tuberculosis was killed in milk
by a heating at 140° F. for 15 minutes. Studies?® were im-
mediately begun at Wisconsin which showed that under com-
mercial conditions the germs in question were killed by ex-
posure to 140°F. for 10 to 15 minutes thus substantiating the
findings of Smith. It was recommended that in commercial
plants the heating be continued for 30 minutes at 140°F.
in order to provide a satisfactory margin of safety. This
method of pasteurization has been found satisfactory from
the commercial point of view and has become the standard
procedure in protecting the milk supplies of the country.

16 See footnote 8.

1747. L Russell, The History of w Tuberculous Herd of Cows, Bulletin
78, Wis. Agr. Exp. Sta., 1899.

1%. LL, Russell, Pasteurization of Milk and Cream for Direct Con-
sumption, Bulletin 44, Wis. Agr. Exp. Sta., 1895.

19 Theobald Smith, The Thermal Death-point of Tubercle Bacilli in Milk
and Other Fluids in Jour. Exp. Med., 4, pp. 217-233, 1899.

2» FW. LL, Russell and E. G. Hastings, Thermal Death-point of Tubercle
Bacilli under Commercial Conditions in Annual Report, Wis. Agr. Exp. Sta.,
17 (1900), pp. 147-170, 1900.

36 UNIVERSITY OF WISCONSIN STUDIES

The process of pasteurization not only frees milk from the
danger of transmitting tuberculosis but at the same time it
frees it from the danger of transmitting the other diseases
which have been observed to have been occasionally so trans-
ferred. Accordingly pasteurization is coming to be recog-
nized as the simplest and most efficient protection against the
spread of disease germs through milk supplies.

THE PROBLEM OF CLEANLINESS

Even under most favorable conditions it is difficult to pre-
vent limited amounts of foreign matter from getting into milk.
On the other hand, the consumer insists that the milk shall
be as clean as practicable. Fortunately the amount of soluble
foreign matter which finds its way into milk is very slight
and the white color of the milk forms a background against
which any insoluble matter present stands out distinctly.

The amount of insoluble dirt actually present in milk as it
ix delivered to the consumer naturally varies considerably.”
The present available data suggest that it rarely amounts to
more than five milligrams per quart or roughly five parts per
million while the average is nearer one part per million.

The Wisconsin Sediment Tester? is a convenient device for
collecting upon a cotton disk the insoluble dirt from a pint
of milk and has proven a satisfactory instrument for this
purpose. At the Chicago Department of Health this method
has been made ronghly quantitative by preparing test disks
from samples of milk containing weighed amounts of dirt.”
At the University of Illinois the standards for quantitative
examinations of sediment in milk have been still further
perfected until it is believed that the weight of the insoluble
dirt in milk may be quickly determined with an accuracy
rivaling that obtained by any other available means.

The standards for this purpose are prepared by powdering

“CH, N. Parker, City Milk Supply, p. 256.

#S. M. Babcock and E. H. Farrington, New and Improved Tests of Dairy
Products, Bulletin 195, Wis. Agr. Exp. Sta., 1910.

38, O. Tonney, Report of the Municipal Laboratory, Department of
Health, City of Chicago, 1907-1910, p. 25.

DEVELOPMENT OF CITY MILK SUPPLY PROBLEMS 37

carbon and passing it through a 100-mesh to the inch sieve.
The desired amount of this carbon is then weighed in a ground
glass slide and transferred to a small beaker containing 50
ce. ¢c. of milk. After stirring until uniformly distributed, the
contents of the beaker, while rotating, are poured quickly into
the sediment tester. The carbon is deposited upon the cotton
disk in the tester and this disk may be used as a standard
for comparison with disks resulting from the testing of milk
samples.”4

Standards prepared in this way are quite uniform and in
the lower dilutions clearly show differences of 0.25 milligram.
A series of these standards may be conveniently mounted on
a card and protected from dirt by a strip of glass or celluloid.
With such a series of standards at hand, the making of a sedi-
ment test from a sample of milk and the estimation of the
amount of dirt present by comparison with these standards
occupies but two or three minutes.

Tur PRoBLEM or KEEPING QUALITY

The first article prepared by Dr. Russell for his first annual
report was entitled The Source of Bacterial Infection and the
Relation of the Same to the Keeping Qualities of Milk.> At
that time the idea was almost universal that the overshadow-
ing importance of bacteria in milk lay in their relation to
the health of the consumer. A portion of this fear of germ
life arose from the feeling that since certain germs cause
definite diseases, all germs must be dangerous. The faulty
logic in this reasoning is too evident to need discussion. The
nightshade is truly a poisonous plant while its near relative,
the potato, is one of our most important sources of food.

The fact that the death rate of babies is highest during the
hot months when the germ content of the milk supply is also
highest early led to the belief that the high germ content

* The details of the preparation of these standards were worked out by
James D. Brew under the general direction of the author.

2H. L. Russell, The Source of Bacterial Infection and the Relation of
the Same to the Keeping Qualities of Milk in Annual Report, Wis. Agr.
Exp. Sta., 11 (1894), pp. 150-165, 1895.

38 UNIVERSITY OF WISCONSIN STUDIES

of milk was responsible for the death of the babies. However,
the results from the most careful studies of this subject fail
to substantiate this view. Park,2* in studying conditions in
New York, found that while under tenement house conditions
high germ content milk and fatalities among the babies were
commonly associated there were likewise present many other
factors inimical to the health of the baby. On the other hand
in baby hospitals where the germ content of the milk supply
was uniformly high but good general care was given the
babies, their health was satisfactory. Williams?’ later made
a careful study of all baby deaths during one year in Roches-
ter, N. Y. He concluded that the milk supply could be
directly connected with only a small portion of such deaths.
Price,”® by instructing the mothers, reduced the baby death
rate in Detroit to a low level without changing the character
of the milk supply.

On the other hand, it is generally agreed that bottle-fed
babies are prone to digestive disturbances which are usually
attributed to something connected with the milk since that is
practically their sole source of nourishment.

It has often been asserted that these digestive troubles were
due to the presence of large numbers of germs in the milk.
On the other hand, when these same ailing infants are placed
upon a diet of milk containing immense numbers of germs,
their digestive disturbances usually promptly vanish. This
would seem to dispose of the argument that their original
trouble was duc to the mere presence in milk of large numbers
of germs.

Logically, the next suggestion is that the difficulty with
the babies is due, not to the germs themselves, but to the
changes which the germs bring about in the milk. The

2... H. Park and L. E. Holt, Report upon the Results with Different
Kinds of Pure and Impure Milk in Infant Feeding in Tenement Houses
and Institutions in New York City, Dept. of Health, City of N. Y., Am.
Rept. (1902), pp, 275-299, 1904.

7 J. R. Williams, A Study of Infant Mortality in Rochester. The Rela-
lion of Market Milk Thereto in N. Y. Med. Jour., July 18, 1912.

*“W. H. Price, Some Statistics Regarding Infant Mortality. in Aw.
Repl,, Int. Asso, Dairy and Milk Inspectors, 3, pp. 95-108, 1915.

DEVELOPMENT OF CITY MILK SUPPLY PROBLEMS 39

marked improvement in the health of ailing infants when fed
upon sour milk is commonly attributed to a difference in the
kinds of germs present in the milk consumed before and after
the improvement with a corresponding difference in the
character of the changes produced in the milk by these germs.

So far as studies have been made, there is shown surpris-
ingly little qualitative difference in the bacterial flora of the
ordinary public milk supply when carrying a high germ con-
tent and the flora of the milk which has acknowledged ther-
apeutic value in the treatment of sick babies. There are, of
course, quantitative differences in that the total number of
germs in the therapeutic milk is much higher and this in-
erease in numbers is largely in germs with marked acid-form-
ing tendencies.

Too little is yet known of the chemical products of bacterial
action in milk to justify conclusions, but the known ability
of milk compounds to absorb or otherwise neutralize small
amounts of bacterial by-products suggests that many of them
may be cared for in this way. Acid is the most evident by-
product of this bacterial action but this is most abundant in
the therapeutic milks.

Failing to find a satisfactory explanation for the illness of
children in the number of germs involved or in any of their
recognizable by-products, it seems logical to seek for other
explanations of the constantly observed coincidence of high
germ content milk supplies and baby sickness.

A part of the diarrheal diseases of children is undoubtedly
due to the action of specific dysentery organisms. No tech-
nique is available by which the presence of these organisms
in milk can be readily determined but pasteurization offers
the same protection against them as against other pathogenic
organisms.

Experience in the feeding of domestic animals has abun-
dantly demonstrated that an animal accustomed to a uniform
dict is markedly disturbed by sudden changes in its ration.
It ig an axiom in the feeding of dairy cows that changes in
the amount or character of their ration should be made
gradually. The gratifying improvements which have followed

40 UNIVERSITY OF WISCONSIN STUDIES

the district nurse system of instructing mothers in uniform
and regular feeding of babies suggests that babies respond
similarly to like physiological stimuli. In the feeding of calves
it is observed that calves like the human infant thrive when
fed uniformly upon sweet milk or when fed uniformly upon
sour milk but that diarrhea appears promptly when a system
of feeding sweet milk is varied by an occasional feeding of
sour milk. While frankly admitting the danger of reasoning
by analogy, the similarity of the factors observed and of the
results obtained in the feeding of calves and of babies strongly
suggests that in both cases diarrheal conditions result from
sudden changes in the acid reaction of the diet. If this rea-
soning is correct, a satisfactory supply of baby milk is char-
acterized among other things by a constant acid reaction.
Under ordinary conditions the reaction which will be most
satisfactory is approximate neutrality.

If the above explanation of the evil results attending the
use of occasional samples of partially sour milk is accepted, it ©
offers an excellent reason for insisting that the public milk
supply shall have such a keeping quality as to maintain an
essentially unchanged reaction during the time which would
ordinarily elapse before its consumption.

Since the increase in acid reaction of milk is due to the
growth of bacteria, the problems of keeping quality are prob-
lems of limiting the entrance and activity of germ life and
of measuring the activity of the germs which enter.

The work of Dr. Russell, above referred to,2° is one of the
earliest inquiries as to the source of the bacteria which enter
the milk and this type of inquiry has been continued by a
number of his students. In connection with this series of
studies it has developed that the barn and barn conditions
exert surprisingly little influence upon the germ content of
milk.*° While in a few instances the udder flora heavily

See footnote 25.

HH. A. Harding, G. L. Ruehle, J. K. Wilson and G. A. Smith, The Effect
of Certain Dairy Operations upon the Germ Content of the Milk, Bulletin
365, N. Y. Asr. Exp. Sta., 1913. ,

M. J. Prucha and H. M. Weeter, Germ Content of Milk as Influenced
by the Factors at the Barn, Bulletin 199, Tl, Agr. Exp. Sta t9i7.

i

DEVELOPMENT OF CITY MILK SUPPLY PROBLEMS 41

infect the milk,*t under ordinary conditions the contamina-
tion of the milk arises almost exclusively from the utensils
in which it is handled.2?, Among the utensils the milking
machine,** where it is used, the shipping can, and the cooling
and bottling machinery at the milk plant are ordinarily the
principal sources of this contamination.

The problem of a suitable test for the keeping quality of
milk is not a3 simple as might at first appear.

Since the keeping quality of milk depends upon germ life,
the number of germs present has been suggested as the logical
measure of keeping quality, and this idea has been accepted
by a considerable number of municipalities. However, there
seems to be no basis for an agreement as to the maximum
number of bacteria to be used as a standard of proper keeping
quality. Actual municipal standards vary from as low as
50,000 per ec. c.** to as high as 2,000,000 per c. ¢.**

A very practical difficuity with bacterial standards is the
extreme variations which occur in determining the germ
content of milk even under the most favorable conditions.
This was most strikingly illustrated in a comparative test
made under the direction of Dr. H. W. Conn and conducted
cooperatively in four laboratories in New York City. These
studies showed that occasionally plate determinations made
from milk approximately sour indicated a lower germ content
than other plate determinations made from certified milk,

31K, G. Hastings and C. Hoffman, Bacterial Content of the Milk of Indi-
vidual Animals, Research Bulletin 6, Wis. Agr. Exp. Sta., 1909.

H. A. Harding and J. K. Wilson, A Study of the Udder Flora of Cows,
Tech. Bul. 27, N. Y. Agr. Exp. Sta., 1913.

2M. J. Prucha, H. M. Weeter and W. H. Chambers, Germ Content of
Milk as Influenced by the Utensils, Bulletin 204, Ill. Agr. Exp. Sta., 1918.

%F,. C. Harrison, Machine-drawn Milk Versus Hand-Drawn Milk in
Cent. F. Bak., II. Abt. 5, 183-189, 1899.

E. G. Hastings and C. Hoffman, Bacterial Control of Machine-drawn
and Hand-drawn Milk in Annual Report, Wis. Agr. Exp. Sta., 24 (1907),
214-222. See also Cent. f. Bak. II. Abt. 22, 222-231, 1908.

H. A. Harding, J. K. Wilson and G. A. Smith, Milking Machines: Effect
of Method of Handling on the Germ Content of Milk. Bulletin 317, N. Y.
Agr. Exp. Sta., 1909.

34 Bacteriological Standards for Milk, U. 8S. Public Health Reports, 29,
pp. 1218-1221, 1914.

33 Limit for potable milk set by ordinance at St. Louis, Mo.

42 UNIVERSITY OF WISCONSIN STUDIES

although the real germ content was vastly higher in the sour
milk. It should be noted that this is a comparison of the
results from individual plates and that the average from a
considerable number of simultancous plate determinations
gave more logical results. A study of the above data by Dr.
Reitz showed that the results of approximately twenty-five
simultaneous plate determinations should be averaged to give
results which are satisfactorily accurate.** Ordinary labora-
tory studies are made with four to six simultaneous plates*’
but routine municipal determinations are frequently based
upon the count from single plates. In the face of the demon-
strated variability of bacterial plate counts, the use of the
results from single plates in municipal laboratories is ex-
tremely unsatisfactory.

Another stumbling block in the way of bacterial plate
standards is the fact that there are so few municipalities
equipped with men and facilities for making as accordant
plate counts as those discussed above. The number of cities
in the United States thus properly equipped is considerably
iess than the number which have already adopted such muni-
cipal standards. Any standard of keeping quality which
is to be a satisfactory aid in measuring the keeping quality
of city milk must be much more widely applicable than
standard bacterial plate counts are at present. The ‘‘little
plate’? method suggested by Frost?* meets some of these ob-
jections but as yet has not been commonly employed.

The direct microscopic estimation of the bacteria in milk
has a number of advantages over the plate count among which
are the quickness with which it can be made and the relatively

*® Data presented by H. L. Reitz and H. A. Harding to the Laboratory
Section of the \mer. Public Health Association at the Rochester meetins,
1914,

“TM. J, Prucha and H. M. Wecter, Bul. 199, Ill. Agr. Exp, Sta., p. 31,
1917.

*“W. D, Frost, Rapid Method of Counting Bactcria in Milk in Sei, 42,
pp. 255-256, 1915; Comparison of a Ranid Method of Counting Bacteria in
Milk with the Standard Plate Method in J. of Inf. Dis., 19, pp. 2738-287,
1916; A Rapid Method of Counting Bacteria in Milk and Other Richly
Seeded Materials in J. Amer. Med. Asso. 66, pp. 889-890, 1916; Counting
the Living Bacteria in Milk—A Practical Test in J. of Bact., 2, pp. 567-
583, 1917: Improved Technique for the Micro or Little Plate Method of
Counting Bacteria in Milk in J. of Inf. Dis.. 28, pp. 176-184, 1921,

DEVELOPMENT OF CITY MILK SUPPLY PROBLEMS 43

small expense for laboratory equipment.®® Among its limita-
tions the fact that it is not readily applicable to milk of low
germ content nor to pasteurized milk are the most important.

As acid formation is the most prominent objectionable
feature in milk of poor keeping quality it has been suggested
as a measure of keeping quality. Studies of acid develop-
ment in milk show that at the temperatures to which milk
is ordinarily exposed during delivery and in the household,
changes in acidity occur at first so slowly as to appear of
little significance but that later acidity imereases very
rapidly.° As a result milk at the time of delivery may be
so little changed as to show on titration a normal acidity and
yet be sour before the lapse of twenty-four hours.

A simple and on the whole fairly satisfactory test of keep-
ing quality may be made by holding a sample of milk at a
definite temperature for a definite time and then examining
for acid and flavors. This test has the merit of not requiring
apparatus more complicated than a good ice chest and facili-
ties for the titration of acidity. It is a test which is within
the financial possibilities of any municipality which has suf-
ficient funds to provide an inspector to collect and observe
milk samples.

Tue PROBLEM OF INSPECTION

Attention has already been drawn to the fact that a city
can learn little about the actual condition of its milk supply
until it provides some form of inspection, but much of the
value of such information depends upon the form of in-
spection provided.

Until about 1900 city milk inspection consisted almost ex-
clusively of the collection and examination of samples within
the municipality. At succeeding periods attention was
focused upon skimming and watering as affecting the food

32, S. Reed and J. D. Brew, Counting Bacteria by Means of the Mi-
croscope, Tech. Bul. 49, N. Y. Agr. Exp. Sta., 1916.

40, G. Hastings and A. C. Evans, 4 Comparison of the Acid Test and
the Rennet Test for Determining the Condition of Milk for the Cheddar
Type of Cheese, Cire. 210, U. S. D. A., Bureau of A. I., 1913.

44 UNIVERSITY OF WISCONSIN STUDIES

value, upon cleanliness and upon the germ content as affecting
keeping quality and also as an index of healthfulness.

After 1900 in milk inspection work attention was shifted
from the city to the country. This shift was made possible
by the development of the dairy score card.** The dairy
score card was originally an attempt to assign such values
to the equipment and methods employed in the dairy that the
resulting score would give a correct index of the general
desirability of the dairy. A dairy scoring 100 would be one
in which all items were ideal. The score of actual dairies
varied greatly but ordinary producing dairies had an average
score of less than 50% which at once suggested that they were
open to considerable improvement.

There is little question but that the quality of milk is deter-
mined by two general factors, heredity and environment; the
former determines the food value of the milk and the latter
controls healthfulness, cleanliness, and keeping quality. The
score cards paid no attention to the food value of the milk
but took account of the environmental factors.

While Cairy svore cards were originally designed as
measures of the general desirability of dairies, as such it is
easy to sce how in the absence of better standards the dairy
score was taken by health officials as an index of the quality
of the milk produced from the dairy. The application of this
new mcans of rating and improving milk supphes seemed so
fascinatingly simple that the New York City Department of
Health was given an initial appropriation of $100,000 with
which to begin the system of farm inspection.

The keen interest in this form of farm inspection lasted
about a devade. During this time it was shown that when the
dairy score became of financial importance to the producer,
either because he was offered a bonus for high scores or be-
cause he would be excluded from the market if his score was

4tWm. C. Woodward, Ann. Rept. of the Health Officer, Dist. of Colum-
bia, 25 (1903-4), p. 27, 1904. Another card independently proposed by
Dr. R. .A. Pearson, Feb. 25, 1905.

42 Geo. M. Whitaker, The Extra Cost of Producing Clean Milk, Ann, Rept.
of Bureau, 26 (1909), pp, 119-131, 1911.

DEVELOPMENT OF CITY MILK SUPPLY PROBLEMS 45

too low, he changed the conditions of production as profound-
ly as financial conditions seemed to warrant. **

The decline in popularity of the score card as the basis for
farm inspection may be ascribed to two general factors. The
first was connected with the matter of germ life in milk.
The period during which interest in the germ content of milk
practically overshadowed other phases of milk quality in the
minds of health officials was practically coincident with the
period of interest in dairy score cards. At the time the score
cards were formulated there was little available information
as to just how bacteria most commonly found their way into
milk. This question soon thereafter received careful study
and unfortunately for the score cards these studies showed
that the avenues through which the great bulk of the germ
life enters the milk*+ had been given slight emphasis on the
score cards. The correctness of this conclusion was shown
by a separate line of inquiry through which it was demon-
strated that there was no observable relation between the germ
life present in the milk of dairies and the score of these dairies
on any of the score cards in general use.** It is entirely con-
ecivable that in the light of added knowledge regarding the
sources of bacteria in milk, new and better score cards might
be formulated.

A second set of facts has militated even more strongly
against farm inspection. Mention has been made of the large
appropriation with which New York City initiated its farm
inspection. However, it soon became evident that with this
appropriation it would not be possible to inspect all the
farms furnishing milk to that city more than once per year.
A survey of the general situation showed that only under ex-
ceptional conditions could funds be made available to provide

“H. A, Harding and J. D. Brew, The Financial Stimulus in City Milk
Production, Bul. 363, N. Y. Agr. Exp. Sta., 1913.

44M. J. Prucha, H. M. Weeter and W. H. Chambers, Germ Content of
Milk. II. As Influenced by the Utensils, Bul. 204, I]. Agr. Exp. Sta., 1918.

J.D. Brew, Milk Quality as Determined by Present Dairy Score Cards,
Bul. 398, N. Y. Agr. Exp. Sta., 1915.

46 UNIVERSITY OF WISCONSIN STUDIES

for more than two farm inspections per year.** On the other
hand the studies of germ life referred to above have shown
that the factors controlling the germ content of the milk
change twice per day on each farm. Under such cireum-
stances little can be accomplished toward controlling the germ
content of milk by the amount of farm inspection which the
health officials find to be financially possible.

The need of effective supervision of milk supples is unques-
tioned. Farm inspection failing to provide this supervision,
attention is returning to an inspection of the milk itself. As a
matter of fact, if the consumer can assure himself that the milk
as delivered to him is in satisfactory condition, he has very
little interest in the preceding details. The four questions which
he is accustomed to ask regarding a milk are the following :*”

Is it rich? (The problem of food value)
Is it safe? (The problem of healthfulness)
Is it clean? (The problem of cleanliness)
Is it sweet? (The problem of keeping quality)

The housewife commonly estimates the richness of the milk
by the depth of the cream in the neck of the bottle. The
food value of milk cannot be adequately expressed by any
single measurement because in addition to its value as a source
of energy, it has an important relation to growth and health.
However, in comparing the relative food values of two samples
of normal milk, the net calories of energy contained are per-
haps the best basis for comparison. There are at present
available only a few analyses of milk of known purity and
these analyses grouped according to their fat content are
eviven in Table I.

4H. Lloyd, Report of Committec on Cost of Dairy and Milk Inspec-
tion, Ann, Rept., Int. Asso. of Dairy and Milk Insp., 7, 42-47, 1919.

47H. A, Harding, R. S. Reed, W. A. Stocking and E. G. Hastings, What
is Meant by Quality in Milk. Circ. 205, Ill. Aer. Exp. Sta., 1919. Also
published by the N. Y. and Cornell Agr, Exp. Stations.

DEVELOPMENT OF CITY MILK SUPPLY PROBLEMS 47

TABLE I.—ENERGY VALUES OF MILKS 48

Carbo-

; Food Calories Calories Totas
Protein Fat hydrates | substance per per calories
per quart* gram quart per quart
1. 2 3 4 5 6 te
per cent per cent per cent grams calories calories calories

2.648 25.87 4 103.48
3.00 29.31 9 263.79

4.596 44.90 4 179.60 546.87
3.068 29.96 4 119.84
3.498 34.18 9 307.62

4.908 47.90 4 191.60 619.06
3.044 29.74 4 118.96
3.994 39.02 9 351.18

4.875 47.62 4 190.52 660.66
2.082 30.11 4 120.44
4.516 44.12 9 397.08

4.958 45.44 4 193.76 711.28
3.62 35.37 4 141.48
5.048 49.32 9 443.88

4.922 48.09 4 192.36 77.72
3.7413 36.57 4 145.28
5.534 54.07 9 486.63

4.93 48.17 4 192.68 825.59
3.992 39.00 4 156.00
5.94 58.03 9 522.27

4.878 47.66 4 190.64 868.91
4.12 40.25 4 161.00
6.50 63.52 9 571.68

4.90 47.87 4 191.48 924.16
4.22 41.23 4 164.92
7.00 68.39 9 615.51

4.84 47.29 4 189.16 969.59

*In computing these values, 977 grams have been used as the weight of one quart
of milk. This is the weight of a quart of milk of specific gravity 1.0323, or nearly
the average specific gravity of milk. The use of the minimum or maximum limits
of specific gravity of normal milk (1.029-1.085) would change the values so slightly
as to be negligible in so far as the purpose of this circular is concerned.

The figures given in Table I show that accompanying an
increase of fat from 3 per cent to 7 per cent, there is a cor-
responding increase in net calorific value from 546.87 to

40, R. Overman, Food Values and Dairy Products, Cire. 235, Ill. Agr.
Exp. Sta., 1919.

48 UNIVERSITY OF WISCONSIN STUDIES

969.59 calories or an increase of approximately 105 calories
with each increase of 1 per cent fat. The use of the calo-
rimeter, however, is too technical even for inspection work,
From the above data it is evident that the use by the house-
keeper of the fat content as an index of food value is correct
in principle though the method of estimation is necessarily
crude. The Babcock test for fat determination is both simple
and accurate with the added advantage that the test itself
is well understood and the necessary apparatus is ordinarily
at hand. Accordingly, the fat content is in all particulars
well adapted to serve as an index of food value of milk as it
is delivered to the consumer.

The safety of the milk as delivered may be about equally
well guaranteed in either of two ways; by frequent inspection
of the health of the cows and people concerned in its pro-
duction and handling, or by proper pasteurization and pro-
tection from reinfection. Safeguarding the milk by means
of health inspection of cattle and men adds from 3 to 5 cents
per quart to the cost of the milk while the expense of pasteur-
ization varies from about 1 cent per quart in small quantities
to about 1, cent per quart in laree quantities. Under such
conditions it is not strange that practically all the actual
advance in safeguarding municipal milk supplies has been
made through the adoption of pasteurization. Where pasteur-
ization is relied upon to make milk safe from germs of in-
fectious diseases, there should be a requirement of tempera-
ture recording devices in connection with the process and a
freyuent inspection to ascertain that the apparatus is in
proper working condition and that the milk is properly pro-
tected after pasteurization.

The housewife is aware that when a bottle of milk is un-
disturbed for some time the dirt in the milk settles to the
bottom and she accordingly looks at the bottom of the bottle
to determine the cleanliness of the milk, In inspection work
the amount of insoluble dirt in milk as delivered to the con-
sumer may be estimated very aceurately by means of the
sediment. test. ‘

DEVELOPMENT OF CITY MILK SUPPLY PROBLEMS 49

There can be no question regarding the relative ease and
accuracy with which the above outlined examinations will give
the facts regarding the food value, freedom from germs of
infectious diseases, and cleanliness of a sample of milk. How-
ever, there is not the same agreement regarding the possibility
of determining the keeping quality. There are at present no
means available for quickly measuring the keeping quality
of milk except in samples which are rapidly approaching an
undesirable condition. In such samples titration of the
acidity may indicate their condition and in the case of un-
pasteurized milk a microscopic examination is even more
useful. However, the results from both such examinations
of some milk samples may seem favorable and still the milk
may not remain sweet for twenty-four hours under household
conditions.

In the absence of any test which will immediately yield the
desired information in the case of pasteurized milk, recourse
may be had either to the bacterial count or to the observation
of a sample of the milk itself. Since the question at issue is
the ability of the milk to remain sweet and in satisfactory
condition for twenty-four or at most forty-eight hours, this
fact can be determined from the sample of the milk itself
quite as quickly as the bacterial count can be determined from
the standard plates. The examination of the sample has the
added advantage that it does not require as extensive equip-
ment nor as delicate manipulation. Most important of all,
it gives precisély the information desired while the bacterial
count even when accurately determined must first be trans-
lated before it can be of service.

Following the plan above outlined it is practicable to collect
samples of the milk as delivered to the consumer and from
the examination of such samples, supplemented by some
inspection of pasteurization plants, determine the food value,
healthfulness, cleanliness, and keeping quality of the milk
supply.

50 UNIVERSITY OF WISCONSIN STUDIES

GrapvIng MILK

An accurate answer regarding the richness, safety, cleanli-
ness, and sweetness of a sample of milk is the real beginning
of successful milk inspection. However, inspection presup-
poses the existence of standards of quality, and in every other
line of merchandise these standards are arranged so as to
recognize classes or grades corresponding to market demands,

The idea of grading milk is not a new one, as in 1866 in
England*® regulations were promulgated recognizing two
grades of milk, one adapted to city trade and one to manu-
facturing purposes. Suggestions for grading the city milk
supply began to be made in New York City before 1907 but
the first official grading of a municipal milk supply in this
country was probably that at Geneva, N. Y. in 1907.°° A
plan of grading the milk supply of New York City went into
effect in January, 1912.°1. This plan established a number
of commercial grades and required that each bottle of milk
bear the designation of its grade. The Milk Commission
appointed by a philanthropic organization called the New
York Milk Committee in 1912 °° recommended a plan for milk
grading which was essentially an amplification of the pre-
vious New York City plan. The grading plans of the city
of New York and of the Milk Commission have been based
mainly upon the results obtained from dairy scores and the
bacterial count of the milk. It should also be noted that
both of these plans for grading recognize the importance of
pasteurization. As already explained no relation has been
demonstrated between the dairy scores as actually obtained
and the quality of the milk while bacterial counts are at
best only an indirect way of measuring the keeping quality
of the milk. The city of New York has continued with its

4H. N. Parker, City Milk Supply, p. 370.

SoH. A. Harding, Publicity and Payment Bases on Quality as Factors in
Improving a City Milk Supply, Bul. 337, N. Y. Agr. Exp. Sta., 1911.

aW. H. Park, The Future Milk Supply of New York City, Ann. Proc.
Amer. Asso. Med, Milk Com., 5, pp. 99-108, 1911.

°2 First Report of the Commission on Milk Standards Appointed by the
New York Milk Committee, Public Health Reports 27, pp. 673-700, 1912.

DEVELOPMENT OF CITY MILK SUPPLY PROBLEMS 51

experiments in milk grading, and in 1913 milk grading was
extended to include the state of New York. Encouraged by
these attempts milk grading has been undertaken by munici-
pal and private enterprise in various cities in a number of
states.

In 1912 the Official Dairy Instructors Association or, as it
is now called, the American Dairy Science Association,
through a committce, undertook to determine the essential
facts in milk quality. In its first formal report in 1917,°*
this committee defined quality in milk and indicated how
grades might be so-constituted as to conform to market needs.

The markets in the large cities and in the smaller towns
present somewhat different problems. In both, the main need
is for a moderately rich, safe, clean, sweet milk which will be
satisfactory for adults and for children. This may well be
characterized as ‘‘table milk.’’ In both there is a limited de-
mand for an extra attractive grade of milk which is usually
somewhat richer, perhaps a little cleaner, and has an increased
keeping quality. This may be referred to as ‘‘special’’ or
“baby milk.’’

On the other hand there is on both markets a considerable
amount of milk which does not come up to the standard for
‘table milk.’’ In the large cities its most common deficiency
is keeping quality induced by age or careless handling. In
the small town healthfulness is usually the point to be criti-
cised since in such places pasteurization is the exception rather
than the rule. Such milk is either undesirable or unsafe,
depending upon the nature of its deficiency, and still in either
case it is ordinarily well adapted to cooking purposes. Such
milk might be characterized as ‘‘cooking milk.’’

It is common for milk reformers to insist that any system
of classification which contemplates the sale of unsafe milk
is unsound but any workable system of classification must
take account of market conditions as they exist. Again, cook-
ing milk as here discussed is safe for cooking purposes. Any
plan which contemplates accurate labeling is at least a step

% See footnote 47.

52 UNIVERSITY OF WISCONSIN STUDIES

in the right direction, and as promptly as the local situation
makes this possible the objection can be met by an ordinance
requiring the proper pasteurization of all of the local milk
supply. Until this point in the development is reached, the
situation is not made any safer where unsafe milk is sold
without a label than it would be if the unsafe milk were ex-
pressly labeled ‘‘cooking milk.’’ On the other hand, in such
communities the proper use of the ‘‘table milk’’ label would
guide at least the most intelligent to the use of a safe supply
and permit the authorities to indicate to the public that
‘‘eookine milk’’ was not safe for general use.

«As an illustration of the way the qualities of food value,
healthfulness, cleanliness, and keeping quality may be used
in establishing grades of milk the following is quoted from
‘an ordinance adopted by the city of Champaign, Illinois, May
191s.

GRADING OF MILK

Section 16. On and after June 1st, 1918, all milk sold in
bottles in the City of Champaign must state on its label its com-
mercial grade and must be true to grade according to the follow-
ing definition of grade, provided that nothing in this ordinance
shall be construed as applying to certified milk as supervised by
the Champaign County Medical Milk Commission.

Two grades of milk shall be recognized, viz.: iable milk and
cooking milk.

TABLE MILK

Table milk as used in this ordinance is defined as milk having
the following qualities:

Food Value: Butter fat content at least 3 per cent. A higher
butter fat content may be stated upon the container. When s0
stated it must be as indicated upon the container. (A reason-
able variation or tolerance from the statement on the container
will be permitted, provided the fat content is at least 3 per cent.)

Healthfulness: As resulting from pasteurization at 140 to
145 degrees Fahrenheit for thirty minutes or from a physical
and tuberculin test of the cattle, and a medical examination of
those who come in contact with the milk during its production.
The tuberculin test and the medical examinations must be made
by a person or persons approved by the Health Officer. Complete

DEVELOPMENT OF CITY MILK SUPPLY PROBLEMS 53

records of all tuberculin tests and medical examinations must be
promptly filed with the Health Officer. Cows must not be ad-
mitted to the herd until after they have passed the physical ex-
amination and tuberculin test. Tuberculin tests of the herd
must be repeated annually and wherever any animals re-act
they must be promptly removed from the herd and in such cases
the tuberculin test be repeated within an interval of- six (6)
months.

Cleanliness: As indicated bv a sediment test showing not
more than 3 milligrams per pint. The amount of sediment to be
determined by comparison with a scale formed by passing
through a cotton filter, milk in which the designated amount of
powdered carbon has been suspended.

Keeping Qualities: Sufficient to remain sweet and in a satis-
factory condition for 24 hours at 60 degrees Fahrenheit, as de-
livered to the consumer,

COOKING MILK

Cooking milk as used in this ordinance is defined as milk
having the following qualities:

Food Value: Butter fat content at least 2 percent. A higher
butter fat content may be stated upon the container. When so
stated it must be as indicated upon the container. (A reason-
able variation or tolerance from the statement on the container
will be permitted, provided the fat content ig at least 3 per cent.)

Cleanliness: As indicated by a sediment test of not more than
6 milligrams to the pint. The content to be determined by a
comparison with a scale formed by passing through a cotton
filter, milk in which the designated amount of powdered carbon
has been suspended.

Keeping Qualities: Sufficient to remain sweet and in a satis-
factory condition for twenty-four (24) hours at 60 degrees
Fahrenheit, as delivered to the consumer.

This ordinance is not suggested as a model but rather as
an illustration of the adaptation of milk grading to a city
where the authorities are desirous of indicating the safe milk
upon the general market and at the same time are unwilling
to require that all the milk be made safe.

Today city milk supplies utilize approximately one-half of
the milk produced in the United States. The problems con-
nected with city milk include a large part of those con-
nected with the dairy and its products.

54 UNIVERSITY OF WISCONSIN STUDIES

When Dr. Russell entered upon his duties as bacteriologist
to the Wisconsin Agricultural Experiment Station in 1893
practically all of these problems were unsolved. During the
succeeding quarter of a century, the rapid growth of the
cities has demanded a growing milk supply and the problems
have arisen in rapid suecession. This Pioneer Bacteriologist
in person and through his students has taken an honorable
part in the solution of these problems.

THE RESISTANCE OF MOLD SPORES TO THE
ACTION OF SUNLIGHT

JOHN WEINZIRL

The present paper is one of a serie§ on the action of light
on microorganisms, and grew directly out of the work on
the action of light on bacterial spores. In that paper it was
shown that bacterial spores resist the action of direct sun-
light from two to eight hours, i. e., they show about sixty times
the resistance of non-spore-bearing bacteria. Having found
bacterial spores to be highly resistant, it was quite natural
to inquire how this resistance compares with that of mold
spores. The simple method devised for making the exposures
has not been tried on mold spores so far as the writer is aware
and it seemed worth while to extend it into this field. The
method has previously been described at length;? briefly
stated, it consists in first making a suspension of the spores
in water or physiological salt solution; from this suspension
a droplet is spread upon a small slip of sterile paper (1 x 3
em.) contained in a sterile petri dish; for this purpose the
standard platinum loop used by bacteriologists works very
well; the infected paper slips are first dried, then exposed to
the sun in a petri dish without the presence of any medium.
This avoids all disturbing factors such as germination, forma-
tion of disinfectants, and the excessive absorption of the chem-
ical rays by the medium. The plates are exposed on wire trays
raised about 16 inches above the floor, and are held so that
the sun’s rays fall as nearly vertically as practicable. After
being exposed a given length of time the inoculated slips are
transferred by means of a sterile forceps to a suitable medium,
nutrient agar being very satisfactory. If the surface of the
medium is dry, then it is well to add a few drops of bouillon.

1American Journal of Public Health, 4, 11, p. 969, 1914.
2 Journal Infectious Diseases, Sup. No, 3, p. 128, 1907.

56 UNIVERSITY OF WISCONSIN STUDIES

For the work with molds an agar containing the nutrient salts,
potato broth, and cane sugar gives excellent growths, although
ordinary nutrient agar serves nearly as well.

For obtaining the spores it is well to use a culture a month
or two old. Controls were always employed to determine
whether the inoculations were successful. Two controls, one
made at the beginning and the other one at the end of the ex-
periment, were sometimes made but this is scarcely necessary
for mold spores possess a remarkable resistance against desicca-
tion.

CuLtTuRES UsEp

Representatives of the more common molds were employed
in the trials. A black mold of as yet undetermined species
represented the Mucoraceae; Aspergillus niger, Aspergil-
lus fumigatus, Aspergillus nidulans, Oidium lactis and
Penicillium glaucwim were the other forms used. All the
forms are exceedingly common and were obtained from the
air or from decayed fruits, excepting the Otdiwm which was
isolated from milk. Aspergillus fuinigatus was included be-
cause of its pathogenic powers for animals.

The following tables present the results when the spores
of these molds were exposed to direct sunlight.

‘TABLE I.—MUCOR SP.

| Time Exposed (hours)
Date | Age of Culture

Growth No growth

Mareh 27, 1915. Se ee a ae ee Diy Shy Be
JUDE 285. OIG, we ws wendy seats Taye 107. ** 2, 4, 6G, 10

RESISTANCE OF MOLD SPORES TO SUNLIGHT

57

TABLE II.—ASPERGILLUS NIGER

| Time Exposed (hours)
Date Age of Culture .
| Growth No growth
SMMG: 21, AQIS visreccraieciee a senretamioe are 60 days......... |
JUNE Q8's5 WIG: cares. oo each sais Ge OS crete deaje
PRATIOS 2G), LOU Dis casita yess, See. tpayhdht yee ovacesetae GS FP ayes trees
PUY 94), WS cance ine or eens aes 13. ee mgieles
NTT YS LU “TOTS scree atece caiscetese scnisiens Ne esa at chase
AML, VB “LOT Dias at Sade aye dade eure aaeee tal Be OE aden eecedes
TABLE III.—ASPERGILLUS FUMIGATUS
|
1 Time Exposed (hours)
Date Age of Culture |
Growth No growth
June 21, 60 dayS......... Dg SON Gecscescrseortcedvestee eciienn Sear rar ine ere S
June 28, Gy Genre Dy Ag Gy WO senearessl| paantarnreenrs 5
June 29, of O8F Eo aoe os | 95 Uy. 1 AS icoxeeull svat sae ae
July 4, HT op.7 > Sonesta BD aise eccianee pas 18, 27
July 10, i: aha ee H WBive cvsucenrtanmed aren 28, 36, 47, 58
July 18, Be SR ashe Sats 1; We Bly. Ae eval eosin we ee:
TABLE IV.—ASPERGILLUS NIDULANS
Time Exposed (hours)
Date Age of Culture
Growth No growth
Maren 227). VOU os ceisiesscene abe eis 17 daySseveeccs Dogs Doig. Bala wicseaccessa | tacts aso Beorandnaontle she
June 28, Se sasaad Sos bis Pe Ay {ic TOiersescrnle te wewenatntorns ee
June 29, ee (oes 6 PGA Re een eee eT CR
July 4, se Bie DB: BG BTecpearstessilie sariencetesacoveeans oem tees
July 10, fs 185 °235; 96y, 475; G8) vonermince de os a9
July 18, es Fe QO DY. 5 1h Da case sso: clbiccasatseantiaynalt SS Se

58 UNIVERSITY OF WISCONSIN STUDIES

TABLE V.—OIDIUM

Time Exposed (hours)

Date Age of Culture
Growth No growth

June 21, 3915
July 10, 1915
July 18, 1915

TABLE VI—PENICILLIUM GLAUCUM

ime Exposed (hours)

Date Age of Culture

Growth No growth

September 30, 1918................ 2
October 25, 1913
October 30, 1913.. i “35
DMLY AG's TOIR. iG 64 crsiciendie daa aisle TS RO esc toltanen

An examination of the above tables shows that the mold
spores possess an extraordinary resistance to direct sunlight.
This resistanee was so unexpected that in the earlier trials
the cultures were exposed for only a few hours; this time was
gradually lengthened until in the last trials the exposure was
continuous for four or five days during the long days of July,
and vet Aspergillus niger and nidulans never failed to grow.
The other species showed some irregularities, but possessed
remarkable endurance. The irregularities may be due to a
failure to infect the paper strips, for in no other way can we
explain the discrepancy in the case of Aspergillus fumigatus
which withstood 42 hours of exposure one time and only 18
hours in an earlier trial.

It is diffieult to explain this extraordinary resistance of
mold spores as compared with bacterial spores which rarely
survive more than five hours. The fact that these spores are
colored suggests that this pigment exerts a protective influence;

RESISTANCE OF MOLD SPORES TO SUNLIGHT 59

doubtless this is true in a measure, but there still remains the
fact that Oidiwm with colorless spores also shows remarkable
resistance.

The fact that mold spores are relatively lighter than other
spores would suggest that they have succeeded better in elim-
inating water from their protoplasm and hence are better able
to resist adverse agents. How far this may really enter into
the explanation it is difficult to say.

The results help to explain the prevalence of mold spores
in air where they greatly preponderate over bacterial spores.
While bacterial spores can resist the action of sunlight but a
few hours, mold spores can resist this action for days. Indeed,
it is doubtful whether sunlight is able to kill mold spores.

SUMMARY

Spores of the following molds were exposed to direct sun-
light on paper slips in glass petri dishes: Mucor Asper-
gillus niger, Aspergillus fumigatus, Aspergillus nidulans,
Oidium, Penicillium glaucum. The results show that mold
spores are able to withstand 58 hours or five days of continual
exposure to the intense rays of a summer sun.

Apparently, sunlight possesses slight, if any, germicidal
powers upon mold spores. This fact helps to explain the
greater prevalence of mold spores than bacterial spores in
air, for the latter are killed usually within five hours.

THE INFLUENCE OF THE PLANE OF NUTRITION
UPON THE PERCENTAGE OF FAT IN MILK
AND THE PROPERTIES OF THE FAT

C. H. Eckues

It is a well-known fact that the percentage of fat present
in milk is subject to wide variations. ‘Among the most im-
portant causes known to be responsible for these variations
are breed of animal, stage of lactation and the individuality
of the animal dependent upon heredity. In addition the
author has found ¢ertain other factors not previously recog-
nized to be of considerable importance. Among these are
seasonal variations,’ fatness of the cow at parturition,’ and
plane of nutrition." Although -the second and third factors
are not identical, they are closely related and some material
concerning both is included in the present paper.

The fat percentage of milk may be increased decidedly
above the normal for the animal concerned for a short time
after parturition, and imereased to a less extent for a longer
period, provided the animal has been fed on a high plane
of nutrition for a sufficient time previous to parturition to
make it possible for a large amount of fat to be deposited in
the body tissues and that the plane of nutrition following
parturition is reduced to a point below the actual requirements
ot the body. The underfeeding following parturition does
not necessarily result from feed being purposely withheld
by the feeder but because the animal when in high flesh at the

1C, H. Eckles, Jahrezeitliche Schwankugen des prozentischen Fettge-
haltes in Kuhmilch in Milchwirtschaftliches Zentralblatt, Heft 11, pp.
488-502, 1909.

4 Ibid., Influence of Fatness of Cow at Parturition on Per Cent of Fat in
Milk, Bulletin 100, Mo. Agr. Exp. Station, 1912.

°C. H. Eckles and L. S, Palmer, Influence of the Plane of Nutrition of
the Cow Upon the Composition and Properties of Milk and Butter Fat.
Overfeeding, Research Bulletin 24, p. 35; Underfeeding, Bulletin 25, P.
106, Mo. Agri. Exp. Station, 1916.

INFLUENCE OF THE PLANE OF NUTRITION UPON MILK 61

beginning of lactation does not appear to be able to consume
sufficient feed to supply the heavy demands upon her body.

Our attention was first attracted to this factor by the
results of an experiment carried out for another purpose.
A Jersey cow was fed very liberally with grain for several
months before calving in order to have her excessively fat.
Immediately after parturition she was put upon a ration,
estimated from feeding standards to be sufficient to maintain
her body weight allowing nothing for milk production. The
surprising part of the result was that the cow continued to
give practically the same amount of milk, 18-24 lbs. a day
for the thirty days she was kept on this ration. The con-
tinued production of milk on a ration sufficient only for
maintaining the body strongly supports the theory now
generally accepted that milk secretion, at least in the early
stages of lactation, is the result of the action of a hormone
which stimulates the udder into activity. The nutrients
necessary for the milk production unquestionably were taken
from her body as evidenced by a decline in weight of nearly
four pounds daily.

The point that especially attracted our attention, however,
was the fact that the fat percentage in the milk during this
period of underfeeding was abnormally high and that it
declined within twenty-four hours after the ration of the cow.
was increased to a point where sufficient nutrients were
supplied for both maintenance and milk production. These
data are shown in Table I.

It will be noted that the average fat content for the thirty
days of underfeeding was 6.01 per cent while the average
for the entire year, which was normal for the animal, was 4.8
per cent. The experiment was repeated with similar results
with a Holstein cow. The average fat percentage for the
year with this animal was 2.99 and during a period of under-
feeding seven days after calving the fat content was 4.47 per
cent.

In order to secure further data on this subject an experi-
ment was conducted with another Holstein cow. This animal
was fed a very heavy grain ration for over a year resulting

62 UNIVERSITY OF WISCONSIN STUDIES

TABLE I.—EFFECT OF UNDERFEEDING UPON THE PER CENT OF PAT

IN MILK
Jersey Cow No. 20
Days Per cent Yield Grain Alfalfa Weight
after calving fat of milk fed hay fed
Pounds Pounds Pounds Pounds
2 4.37 22.2 3.5 7 830
4 5.80 20.9 3.5 7 807
6 6.89 22.3 8.5 7 790
8 7.21 23.1 3.5 7 187
10 6.60 22.6 3.5 7 785
12 5.86 21.5 3.5 7 780
14 6.82 20.0 3.5 7 765
16 6.00 19.1 3.5 7 755
18 5.07 21.8 3.5 7 755
20 4.94 17.2 3.5 7 730
22 6.37 19.0 3.5 7 730
24 6.82 19.6 3.5 7 725
26 5.70 19.1 3.5 7 720
28 6.26 17.8 3.5 7 720
5.60 19.9 8.5 7 710
32 5,53 20.4 5.5 ll 690
34 4,74 24.4 7 11 710
36 4.28 25.4 7 14 710
38 4.49 25.0 7 lt 720
40 4.45 25.0 7 14 710
42 3.98 27.2 7 14 715
44 4.83 26.7 ve lk 740
46 4.27 26.1 8 16 700
48 3.80 25.9 8 16 735
50 4.23 27.0 8 16 747
DOE alll Sh sreth adsanejadbared 25.6 8 16 725
54 | 4.01 25.7 8 16 715
56 4.47 23.7 8 16 720
58 4.37 24.3 8 16 740

in her being ‘excessively fat at parturition. Beginning ten
days after the birth of the calf, this cow in seven days pro-
duced 291 pounds of milk containing 5.1 per cent of fat. Her
average for the entire milking period of eleven months was
3.2, During the year she was fed a normal ration for a dairy
cow, resulting in her being in a moderate condition only at

the time of next parturition.

Beginning the same interval

after calving as before she produced 224 pounds of milk in
seven days with an average fat percentage of 3.63. The
average for the entire milking period was 3.00 per cent.

INFLUENCE OF THE PLANE OF NUTRITION UPON MILK 63

These results are corroborated by a large number of data from
other experimental animals which will not be given here.

Other evidence on this point is found in the reports of
official tests of dairy cattle as reported by the various breed
associations. All interested in the practical phases of dairy
husbandry are familiar with the immediate application which
followed the publication of the factst regarding the possibility
of increasing the richness of the milk by these means and the
astonishing results that have been obtained in seven day tests
of dairy cows in recent years as a result. It is well known
that the great increase in butterfat yield in these tests is due
largely to a decidedly higher fat percentage. For example
ia 1903 the cow, Sadie Vale Concordia, held the Holstein
butter fat record for seven days with a yield of 694.3 lbs. of
milk containing 3.52 per cent of fat.* The record up to 1917
is 730 lbs. milk containing 5.54 per cent of fat. At the
same time there is no evidence of any appreciable raise in
the average fat percentage for the breed. The average fat
percentage reported by the Holstein Association for the
highest twenty-five cows in seven day tests is 5.1. The aver-
age for the highest twenty-five yearly records is 3.96.

The practical use of this means of greatly increasing the
butterfat production temporarily has resulted in an entirely
new basis of fixing value for breeding stock among certain
of the dairy breeds.

The question may be raised whether it is not normal re-
gardless of the state of flesh of the animal for the fat per-
centage to be high during the first four weeks after parturition
followed by a decline to a lower level. Data are also at hand
showing this is not the case. In Table II are found data giving
the fat content by weeks in the milk of cows that were in a
moderate state of flesh at parturition. It will be noted that
there is a constant rise in the fat percentage from the be-
ginning and that the average for the year is decidedly higher

4C, H. Eckles, 4 New Factor Influencing the Per Cent of Fat in Cows’
Milk in Hoard’s Dairyman, 40, 28, p. 696, Aug., 1909.

6 Holstein Friesian Yearbook, 3, p. 93, 1903.

® Hoard’s Dairyman, 48, 1, p. 14, Jan., 1917.

64 UNIVERSITY OF WISCONSIN STUDIES

than in the beginning of the lactation period which is exactly
the reverse of that found when the animals are in a fat con-
dition,

TABLE II—SHOWING FAT CONTENT NORMAL TO LOW IN BEGINNING
OF LACTATION PERIOD

Cows Moderate to Thin in Flesh

Time No. 57 No. 215 No, 304 No. 59 No. 55 No, 307
after calving

Weeks :
hn eres pene ie)
2 3.0
3 3.2
£ 3.2 3.15
5 4.0 3.1
6 4.4 3.2
i 4.7 3.2
3 4.5 3.0
" 4.2 3.2
10 5.6 3.2
i es 3.2 i
2 4.6 3.30 |

Months
4 5G 3.2 4.0 5.6 4.7 3.30
5 a8 Leal 5.2
6 5.8 3.5 5.3
vi 5.2 3.4 5.3
‘ 5.3 3.6 5.0
9 5.5 238 5.1
10 5.1 3.2
11 5.1 229
5 nn aterener ere es Jed

Year 5.33 3.29 4.01 5.11 lS |aswtiwaenais

We are also able to present data showing the effects of
underfeeding upon the fat percentage are not confined to the
interval immediately following calf birth. Table III gives
the results for Jersey Cow 62 beginning at a point 96 days
in milk. It will be seen that there was almost an immediate
rise in the fat percentage when the ration was reduced below
the requirements and that the fat declined again as soon as
the ration was raised.

INFLUENCE OF THE PLANE OF NUTRITION UPON MILK 65

TABLE III
EFFECT OF UNDERFEEDING—ADVANCED LACTATION

Jersey Cow No. 62

Date Per cent Pounds Live Energy Energy

of fat of milk weight supplied required

Therms herms
4.6 ; 16.9 930 10.00 11.80
4.5 16.3 930 10.00 11.58
4.9 15.6 915 7.00 11.24
4.8 14.6 910 7.00 10.85
5.5 14.8 900 7.00 10.86
5.7 15.1 890 7.00 10.91
5.5 14.6 870 4.66 10.60
5.9 14.8 865 4.66 | 10.43
6.1 13.6 865 4.66 10.21
5.6 12.9 855 4.66 9.88
6.0 13.5. 855 4.66 10.11
6.5 13.1 845 4.66 9.91
5.7 12.9 835 4.66 9.71
6.2 12.9 835 4.66 9.77
5.6 12.9 840 4.66 9.81
5.8 13.3 830 4.66 9.89
5.2 12.8 850 4.66 9.83
5.4 12.0 830 4.66 9.42
5.7 12.8 815 10.00 9.62
4.3 13.1 845 10.00 9.91
4.8 14.5 855 10.00 10.47
4.3 15.2 855 10.00 10.73
4.1 15.3 830 10.00 10.92
4.5 16.1 880 10.00 11.21

Table IV gives similar data for Jersey Cow 2, beginning
100 days after parturition. It will be seen that the results
are the same as for Cow 62. When the ration was dropped
suddenly on the fifth day there was an immediate rise in the
fat content followed by a decline when the ration was again
raised.

Outside the publications of the author, and his co-worker,
Dr. L. S. Palmer, the only reference to be found in the litera-
ture regarding the relation of underfeeding to the fat content
of milk is by Lusk who conducted a brief experiment with
a goat. He reports that the fat content of the milk was in-
creased during two days when the goat received no feed.
In explanation, Lusk advances the theory that when suf-

5

66 UNIVERSITY OF WISCONSIN STUDIES

ficient sugar is not oxidized in the cells the sugar hungry
cells attract fat. Dextrose is converted into milk sugar in
the mammary gland and cannot then be burned in the or-
ganism; the mammary c¢cll becoming a sugar hungry cell
attracts large amounts of fat which is present in the blood.

TABLE IV
EFFECL OF UNDERFEEDING—ADVANCED LACTATION

Jersey Cow No, 2

Date Per cent Pounds Live Energy Energy
of fat of milk weight supplied required
'Therms Therms:
APTA yore essa necaie < 4.8 11.9 870 15.76 9.69
Died cicesoavennise 4.4 11.3 &70 15.76 9.47
Oe 4.5 10.9 870 13,53 9.32
DOngatite@ anes 5.8 11.1 865 10.58 9.36
eM ye eerie se aie 5.4 10.6 842 9.07 9.05
Dia rotate 5.5 10.7 840 &.32 9.07
DR cispeadinettes deo 5.4 10.8 855 9.47 9.20
ON Se as Bieta 5.0 10.9 860 11.27 9.26
Uda alas ces sae 4.7 10.7 852 11.98 9.14
Mays Disccasgs nd tti fs 5.0 13.5 852 15.78 9.79
D Sati siatsactate sa 5.2 Tel 850 15.75 9.27

The author in connection with Dr. L. 8. Palmer has given
some attention to the question of the cause of this increase in
fat content which accompanies underfceding. <A. series
of expcriments was conducted to test a theory that the in-
crease in the fat in the milk is due to an inercase in the fat
in the blood of the animal during underfeeding, a condition
known to occur with smaller animals, at least, under condi-
tions of starvation.

Two cows, a Shorthorn and an Ayrshire, were selected for
the test. These animals were fattened to an excessive point
before parturition in order to make certain that high fat con-
tent of the milk would result after calving. Ten days before
the animals were due to ealve, blood was drawn from the
jugular vein and analyzed for pereentage of fat. Blood sam-
Ties were again taken after parturition when the conditions of
underfeeding were strongest and again later when the animals
were normal. The method of analysis used was the Kama-

INFLUENCE OF THE PLANE OF NUTRITION UPON MILK 67

gaira-Luto as modified by Rosenthal and Trowbridge.’ Table
V gives the results.
TABLE’ V

INFLUENCE OF UNDERFEEDING ON PERCENTAGE OF FAT IN BLOOD AND
PERCENTAGE OF FAL IN MILK

Daily *Pat Fat in Blood
Date Lactation milk in
yield milk Test 1 Test 2. | Average
Days Pounds | Per cent | Per cent | Per cent | Per cent
Cow 407
1914
Dee. 24 SSI thaaiecche tate wae hee etiaats 0.484 0.396 0.440
1915
Jan. 5 3 46.1 6.80 2318 328 323
Jan. 6. 4 33.7 4.80 2S «B15 298
Feb. 16. 45 49.5 3.65 2323 2824 324
|
Cow 305 ;
1915 |
Feb. ee ee ee eee 257 Lost 257
Feb. 2 24.3 6.46 241 223 232
Feb. 3 27.0 6.68 237 e021, 229
Feb. 4 29.8 6.10 +210 Lost 210
Feb. 11 33.1 6.10 +233 Lost 233
Mar. 20 38.0 5200. Psnctcsh accliietece tens leone ees ¢

* The average of Cow 407 for the year was 3.36 and of Cow 305, 4.08 per cent.

The negative results obtained on the blood fat seem to in-
dicate that in the case of the cow, starvation does not result
as is the case with small animals in an increase in the fat in
the blood. It would seem therefore that the increase in the
fat in the milk could hardly be looked upon as the result of
a direct transfer from the body.

It is believed the explanation may be sought along other
lines and the following is suggested. When the body is forced
to draw on its reserves for needed energy, the fat is liberated
by an increased lipase activity. The hormone or chemical
messenger that stimulates this lipase activity must be carried
by the blood, and it is reasonable to believe that it will affect
the lipase activity in all parts of the body through which

7H. Rosenthal and P. F. Trowbridge, The Estimation of Fat in Blood
in J. Biol. Chem., 20, pp. 711-717, 1915.

68 UNIVERSITY OF WISCONSIN STUDIES

the blood passes, particularly those parts in which lipases
are already normally at work. Other enzymes are also in-
volved in this activity, for the synthesis of fatty acids and
glycerol from which the lipases build the neutral fat is
fundamentally reactions of oxidation and reduction. The
general hypothesis, however, is at once seen to offer a plausible
explanation for the increase in the fat of the milk during
underfeeding. It explains how the lipases and other enzymes
normally accelerating the formation of milk fat in the mam-
mary gland may bring about a greater formation of milk fat
at the same time the lipases in the tissue fat of the body are
liberating more fat from the cells for energy required by the
body. It also explains why the highest fat tests in under-
feeding were secured in the cases of the fattest animals, and
why animals in thin or only moderate flesh showed the least
effects on the percentage of fat in the milk.

The relation of the lipase activity of the mammary gland
to the lipase activity in other parts of the body also explains
the low fat percentage of milk often secured during very hot
weather. It is a well-known fact that an abnormally low
percentage of fat in the milk will accompany a period of hot,
humid weather in summer as has been shown by the author,
while a period of very cold, dry weather in winter will cause
an inerease in the percentage of fat in the milk.

INFLUENCE oF PLANE or Nutrition UPon COMPOSITION
Or THE Far

A striking result of underfeeding a lactating cow is the
effect upon the composition of the milk fat. This change
becomes noticeable almost as soon as the plane of nutrition
drops below the requirements of the animal. In the ex-
periment with the Jersey cow mentioned in connection with
the relation of the plane of nutrition to the fat percentage of
the milk, the fat constants exhibited the typical effect of un-
derfeeding during the period when the ration was below the
requirements of the animal. When the ration was increased
to a normal amount the fat constants quickly returned to
normal for the animal in question.

INFLUENCE OF THE PLANE OF NUTRITION UPON MILK 69

At the end of thirty days’ underfeeding the saponification
number was 222:1; after fourteen days on a normal ration it
was 225.7. During the same interval the iodin number de-
creased from 42.62 to 34.15; the Reichert-Meissl increased
from 21.43 to 24.2, and the melting point declined from
36.35 to 33.60.

A summary of the results of twenty-three experiments is
given in Table VI. The plane of nutrition as indicated in
percentage is calculated on the basis of the requirements of
the animal in question calculated according to the Armsby
Feeding Standard. :

The data presented in Table VI have several striking feat-
ures. The most striking is the fact that without exception all

TABLE VI

VARIATION IN FAT CONSTANTS FROM NORMAL TO ABNORMAL DUE TO
PLANE OF NUIRITION

Variations from Normal of
Plane of |—
Exp. Cow nutrition Saponi- Reichert- Iodin Melting
fieation Meissl value point
value number
Per cent ae
20 —62 — 7.2 —5.5 +12.4 +3.5
206 —60 foe eee —9.9 + 8.0 +7.0
301 —39 —15.2 —1.5 +17.5 +2.2
300 —-38 — 3.3 —2.6 + 1.3 +2.2
2 —23 — 5.4 —2.9 + 4.2 +0.3
301 —16 — 4.4 —4.5 + 1.0 +0.8
2 —33 —12.2 —6.9 414.9 +0.7
361 —25 — 79 —4.1 +10.8 —0.8
20 —23 — 2.0 —2.0 + 4.5 +0.0
206 —20 — 2.3 —1.9 + 2.7 0.1
62 AS — 5.8 —1.3 +13.3 —5.0
—30 — 6.7 —3.7 + 7.3 +1.5
300 —21 —10.5 —4.1 +11.6 —2.7
300 —18 — 4.7 4.0 + 6.9 +¢.1
301 —17 —4.1 —3.3 + 5.0 —2.4
300 | —4 — 8.2 —3.2 +10.8 —1.2
DO oie see asbatendeduensgs 301 | +18 to —1.5 — 6.9 —5.0 +°7<.5 —1.0
300 — 9.4 —3.2 + 4.1 —0.2
DOG» Naar ece vals sulin dys setae aie. —4.9 +11.0 +3.5
209 —14.0 —7.0 + 6.3 +6.0
211 —13.8 —&8.2 + 5.8 +8.5
407 — 3.7 —3.3 + 4.5 +2.7
305 — 5.2 —2.0 + 5.2 —1.2

* Subnormal.

70 UNIVERSITY OF WISCONSIN STUDIES

of the experiments show a decline in the saponification value
and Reichert-Meissl number of the butter fat, and an increase
in the iodin-absorption value. The melting point, however,
in some cases was increased, in some cases was not affected,
and in other eases declined. Another striking feature of
the results is the extreme yariations in the chemical fat con-
stants obtained in a great many of the experiments. In
fifteen of the twenty-three experiments given in the tables
the maximum iodin-absorption value rose to a figure of 40.0
or above and in twenty-one of the twenty-three experiments
to a figure of 36.0 or above. The extent of these variations is
emphasized when they are compared with the average normal
iodin value of 32.7 for all the experiments. Similarly,
Reichert-Meissl numbers below 24.0, the legal standard for
butter in the United States, were obtained in ten of the twen-
ty-three experiments. Reichert-Meissl numbers below 28.0, the
Belgian legal standard for butter, were obtained in twenty
of the twenty-three experiments. Again, the extent of these
variations is emphasized by comparison with the average
normal value of 29.1 for the twenty-three experiments.

Application of the results of underfeeding studies. The
riarked variations in the chemical and physical constants
of the milk fat which are found to accompany a subnormal
plane of nutrition, even in cases of mild underfeeding, or
when the plane of nutrition is merely reduced from super-
normal to normal, at once indicate the importance of taking
into account this important factor in both human nutrition
and in experimental work involving the influence of specific
factors on the composition of milk fat.

It is certain that at times milk comes to the market from
herds in such a condition of underfeeding that the fat will
have the somewhat abnormal composition noted in the experi-
mental work reported. It is of interest in this connection
to raise the question as to the possible relation between the
character of the fat and human nutrition, especially of infants.
Unfortunately up to the present no information is at hand
concerning this possible relation althongh it seems entirely
possible it may be a matter of some importance. The im-

INFLUENCE OF THE PLANE OF NUTRITION UPON MILK 71

portance of controlling the plane of nutrition of the
cow becomes especially apparent in studying the influence of
specific feeds on the composition of the milk fat, in which
the changes in the ration which are necessary for studies of
this character may bring about a temporary underfeeding
of the cow. The great care which it has been found necessary
to exert in studies of this character at this Experiment Station
to avoid involving the effects of underfeeding with the effects
of specific feeds throws great doubt on the results of many
of the studies of this kind that have been published in the
past. Not only is it necessary to control this factor in feeding
studies, but it is also necessary to take it into account in the
interpretation of the results. The data secured in the under-
feeding studies are also useful in explaining some of the
heretofore unexplained cases of abnormal butter that are
occasionally reported in the agricultural literature.

Application to feeding experiments. The danger of
allowing the effects of underfeeding to interfere with the
correct interpretation of the effects of specific feeds on the
composition of the milk fat is no more strikingly shown than
in experiments to determine the effects of fresh pasture grass
on the composition and properties of butter fat. Not only is
there danger of a cow in good flow of milk being underfed if
suddenly turned from dry feed to pasture, but more or less
underfeeding is practically certain to follow such a procedure.
This is due to the relatively low nutritive value of fresh
pasture grass together with the fact that the animal is not
accustomed to depend upon her own activity for the feed
which she requires.

An example of underfeeding accompanying a sudden
change from dry feed to pasture is shown in Table VII.
The data were taken at this Station. The animal used was a
pure-bred Jersey weighing about 850 pounds. Her energy
requirement for maintenance and milk production was about
eight therms, which was just supplied by the dry ration.
The animal was turned to pasture for the first time on the
morning of May 20 and thereafter received no additional feed.
The certainty that the animal would be underfed for a few

72 UNIVERSITY OF WISCONSIN STUDIES

days at least is apparent when it is considered that she sud-
denly was required to gather by her own activity about
seventy pounds of pasture grass to supply the eight therms of
energy which she required. Even this figure may be too low,
for it is based on the energy value of green alfalfa, which
is in all probability higher than that of the fresh blue grass
pasture to which the animal was turned and for which no
data are available. The underfeeding results, which were
temporary, are seen in the marked drop in the saponification
and Reichert-Meissl values of the milk fat, and in the great
increase in the iodin value. The fact that underfeeding
accompanied a change to pasture in the case of this cow
with a low milk production emphasizes the danger as well as
the probability of a similar but more pronounced result
accompanying the turning to pasture of cows with much
greater milk production.

TABLE VII

CHANGING THE RATION FROM DRY FEED T0 FRESH PASTURE, SHOWING
THE EFFECT OF UNDERFEEDING *

Milk Fat in Saponi- Reichert- Iodin Melting
yield Date milk fication Meissl value point
value number
Pounds Per cent °C.
18.8 May 18-19 4.55 224.0 25.92 37.02 32.83
4.5 May 20 a. m. 4.35 225.7 24.05 39.11 33.40
4.8 May 21 a. m. 4.47 221.1 23.99 42.68 32.10
4.0 May 21 p. m. 5.76 219.5 22.69 45.34 31.40
5.0 May 22 a. m. 4.04 223.2 “25.10 38.28 33.37
5.7 May 22 p. m. 5.29 223.6 24.68 41.67 32.67
9.8 May 23 5.00 223.6 25.87 43.19 31.87
19.6 May 24-25 4.92 223.7 27.15 42.15 32.27

* Fed on May 18-19, 4.5 pounds of grain, 4.5 pounds of alfalfa hay, and 18
pounds of silage. The other data are based on pasture only.

Equally striking results were obtained in several other
experiments when the ration was grain and dry roughage.
In all these cases the striking change in the properties of the
fat might have been attributed to the change in ration while
the real cause was an insufficient amount of nutrients.

INFLUENCE OF THE PLANE OF NUTRITION UPON MILK 73

It is clear special care must be taken in studying factors
influencing the properties of butter fat that the results are
not vitiated by this factor of under-nutrition. A survey of
the literature of the subject makes it clear that certain in-
vestigators have made this error and the results they attrib-
uted in some cases to certain feeds are really the results of
under-nutrition.

Application to cases of abnormal butter.—Cases of
abnormal butter are not infrequently reported in the agri-
cultural and chemical literature, for which no adequate ex-
planation can be offered. Most of these cases are reported
by food officials whose duty it is to detect adulterated butter,
and accordingly have to do largely with the Reichert-Meissl
number of the butter fat. Such cases are much more fre-
quently reported by officials in European countries than in
the United States. It will not be possible to take up all
of the cases that have been reported. Several typical ones
have been selected, however, and the applications of the re-
sults of the underfeeding studies pointed out in explanation
of the abnormalities found.

Van der Zande® has reported a feeding experiment in which
a decrease in volatile acids occurred while the cows were on
pasture. ‘The change in the composition of the butter fat was
attributed by the author to a period cf severe weather during
the experiment. In view of the experiments pointed out
above showing the ease with which cows may be underfed
while on pasture it seems evident that this factor was re-
sponsible for the change in the composition of butter fat
found by Van der Zande.

Reinsch® has reported a sample of abnormal butter fat from
five Holstein cows, which showed a Reichert-Meissl number of
19.7 and a saponification value of 213.9. The cows were in good
health, and had been in milk but a few months. Their ration
consisted of pasture grass ad libitum with the addition of six

*K. H. M. Van der Zande, Verslag. Ver. Exploit. Proef. Hoorn, pp. 45—
50, 1902.

9A. Reinsch, Ein Fall von sogenannter “anormaler’ Butter in Z. Nahr,
Genussm., 8, pp. 505-508, 19.04.

74 UNIVERSITY OF WISCONSIN STUDIES

to eight pounds of potato pulp and three-fourths to one pound
of rice polishings a head daily. The explanation of the ab-
normally low saponification and Reichert-Meissl values un-
questionably lies in this fact. The conclusion that would
naturally be drawn in the light of the studies of the influence
of underfeeding is that the animals were underfed on account
of the short pasture.

Ziegfeld'!® reported a sudden drop in the Reichert-Meissl
value of the butter fat during a period of feeding cows on
beet-tops, which is attributed to a sudden fall in the tempera-
ture, causing the leaves to become to some extent frozen.
Inasmuch as the cows were fed the beet-tops ad libitum with
no additional feed, it would hardly be expected that they
would consume the required amount to support maintenance
and milk flow if they were suddenly required to eat the partly
frozen beets. A number of other equally clear cases have
been gathered from the literature.

CONCLUSIONS

The percentage of fat in milk ean be influenced to a marked
extent for the first twenty to thirty days by the fatness of the
animal at parturition. This influence appears to extend in
some cases in a less degree for at least three months.

Underfceding of the animal after parturition seems to be
a necessary condition to bring about this abnormal percentage
of fat in the milk.

Tests of. dairy cows made for short intervals in the be-
ginning of the lactation period cannot be depended upon to
indieate the normal percentage of fat produced by the cows
tested.

Underfeeding of the lactating cow results in a decline in
the Reichert-Meiss] number and the saponification value and
an inerease in the jodin value of the milk fat.

The inerease in the percentage of milk fat and the changes
in the character of the fat which accompany underfeeding

1M, Ziegfleld, Die Zusammenseizungd des Butterfetics bei Riibenbdlatt-
fiitterung in Z, Nahr. Genussm., 17, 179-86, 1909.

INFLUENCE OF THI PLANE OF NUTRITION’ UPON MILK 75

are not satisfactorily explained by the hypothesis that under-
feeding causes a transfer of tissue fat to the mammary glands.
Blood fat analyses made during extreme underfeeding in the
ease of two cows failed to show any increase in the amount of
fat carried by the blood stream in comparison with normal
conditions in the same animals. 3

The effects of underfeeding on the composition and proper-
ties of milk and butter fat show the importance of controlling
this factor in feeding experiments involving the effects of
specific feeds on the composition of milk and butter.

The effects of underfeeding must be taken into account
in the interpretation of all data involving variations in the
composition of milk and butter fat due to specific conditions
of the cow, specific environments of the cow, changes in the
feed of the cow, or to feeds of specific character.

CHARACTERISTICS AND DISTRIBUTION OF THE
COLON-AEROGENES GROUP *

L. A. RoGers

INTRODUCTION

The results of a series of investigations carried on in this
laboratory on the gas-forming bacteria, commonly known as
the colon-aerogenes group, have appeared in various journals,
This paper has offered an opportunity to bring these results to-
gether and present them in a briefer and more concise form
t an has been possible in the earlier papers. The results
which will be discussed in this paper represent the accumula-
tive work of a number of persons for which the writer has
no desire to take undue eredit. Particular credit is due my
ceclHeague, Dr. Clark, who has been responsible for the greater

* Published by permission of the Secretary of Agriculture.

1L. A. Rogers, W. M. Clark and B, J. Davis, The Colon Group of Bac-
teria in Jour, Inf. Dis., 14, 8, pp. 411-475, 1914.

L. A. Rogers, W. M. Clark and A. C. Evans, The Characteristics of Bac-
teria of the Colon Type Found in Bovine Feces in Jowr. Inf. Dis., 15, 1,
pp. 99-1238, 1914.

Wm. Mansfield Clark, The Final Hydrogen Ion Concentrations of Cul-
tures of Bacillus Coli in Jour. Biol. Chem., 22, 1, pp. 87-98, 1915.

L. A. Rogers, W. M. Clark and A. C. Evans, The Characteristics of Bac-
teria of the Colon Type Occurring ow Grains in Jour. Inf. Dis., 17, 1,
pp. 137-159, 1915. ;

W. M. Clark and H. A. Lubs, The Differentiation of Bacteria of the
Colon-Aerogenes Family by the Use of Indicators in Jour. Inf, Dis., 17, 1,
pp. 160-173, 1915.

L. A. Rogers, W. M. Clark and H. A. Lubs, The Characteristics of Bac-
teria of the Colon-Aerogenes Type Occurring in Human Feces. (Unpub-
lished. )

S. H. Ayers and P. Rupp, Simultaneous Acid and Alkaline Bacterial Fer-
mentations from Dextrose and the Salts of Organic Acids Respectively.
(Unpublished. )

L. A. Rogers, The Occurrence of Different Types of the Colon-Aerogenes
Group in Water. (Unpublished. )
jen en CHINAS ce rae

: sila t yo pplication in Bacteriology in Jour. Bact,
» J, pp. 1-34; 2, pp. 109-186; 3, pp. 191-286, 1917,

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 77

part of the exceptionally fundamental and searching chemical
investigations on which the bacteriological work has been
based. While intended originally as a study of some of the
gas-forming bacteria of milk, it has developed into an in-
vestigation of the characteristics of the colon-aerogenes group
in general and of the more intimate processes by which some
of the familiar end products are produced. In this way it
has been possible to bring about some semblance of an orderly
arrangement of the large number of cultures studied and to
correlate the types so produced wi.h certain definite habitats
ia nature.

There has been a tendency to arrange bacteria according to
the effect that they have on our bodies or on the food we eat.
This has certain advantages, but is as illogical from a taxo-
nomic standpoint as a classification of men on the basis of their
occupation, an arrangement which would be useful for in:
dustrial purposes, but which would separate members of the
tame family, and bring tcgether distinct ethnological groups.
In the same way bacterial classifications of convenience fre-
quently separate closely related bacteria and bring together
types agreeing only in superficial characters. On the other
hand, there has developed in some quarters a tendency to
follow the logical method of classifying bacteria by deter-
mining actual relationships and lines of descent. This raises
the difficult question of the method of ascertaining how the
evolution of a bacterial species or genus may be traced; in
other words, which characters will be considered as funda-
mental and showing broad relationships, and which are super-
ficial and thus of secondary importance. The record of the
struggle to secure food and opportunity for reproduction is
found especially in msrphology in animals and the higher
plants and in physiological characters in the bacteria and
similar microorganisms.

The colon-aerogenes family is particularly distinguished
by its ability to bring about a gaseous fermentation of carbo-
hydrates, alcohols and glycerines, and in attempting to sep-
arate this extensive family into species, bacteriologists have
usually relied on this fermentative activity as a basis of

78 UNIVERSITY OF WISCONSIN STUDIES

elassifivation. The hopelessness of establishing a stakle classi-
fication by using only the fermentations of various substances
is shown by the large number of proposed schemes, some of
which permit over one hundred possible varieties. The one
which has been most generally followed was proposed by
MacConk+y and is merely the possible combinations of the
plus and minus signs under two test substances. The four
species permitted by this arrangement may, by applying
similar methods, be further divided into varieties, limited
only by the number of test substances used. A classification
of this kind may be convenient in that it adapts itself readily
to a workable key but there is no evidence to show that it has
any relation to the evolution of the group or that it serves
in any way to indicate the origin of the culture.

It should be possible to discover some combination of char-
acters which separates the group into natural species. Since
the evolution of a bacterial species is largely a question of
environment, the species so established should be coincident
with more or less well-defined habitats. For taxonomic pur-
poses it may safely be assumed that the nature of the fer-
mentation is of more significance than the particular material
fermented.

THE CHARACTERS STUDIED

The characters of a group of bacteria can be properly
established only by studying a considerable number of cul-
tures. The value of this method of study has been so com-
pletely demonstrated by some of the recent work on systematic
bacteriology, particularly by the work of the Winslows on the
Coceaceae, that its use should need no further justification.
In attacking this problem we have so far as possible used
exact chemical methods in determining the reactions produced
hy the bacteria in various culture media. This has limited
the number of cultures which could be studied but has per-
mitted distinctions on the basis of quantitative measurements
which have been of the greatest value. The details of the
methods followed have been given in the papers cited and
for the sake of brevity will not be repeated here. The enl-

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 79

tures used consisted of a collection of 689 cultures fermenting
dextrose with evolution of gas, without spore formation,
with abundant growth on agar and diffused growth in broth.
One hundred and fifty-two of these came from 27 samples of
human feces, 148 from 17 samples of bovine feces, 132 from
31 samples of water, 143 from 33 samples of grains, and 114
from milk. They were obtained for the most part by direct
plating on asparagin lactose litmus agar followed by replating
and a test for fermentation in dextrose or lactose broth.

THE FORMATION OF GASES

The active evolution of gases, indicating a more profound
fermentation of the carbohydrates, is the striking character
which distinguishes the colon-aerogenes group from most of
the carbohydrate-fermenting bacteria. The nature and the
relative proportion of gases evolved has come to be regarded
as of little significance and the fermentation tube of Theobald
Smith is now used merely to determine the fact of gas pro-
duction. The gas ratio, proposed by Smith? as a diagnostic
character, has fallen into disrepute through its failure to
correlate with habitat or with other physiological characters.
This attitude is well illustrated by the paper by Longley and
Baton. The probable cause of this failure is found in the
work of Keves* who pointed out the great variation in the
gas ratio as ordinarily determined and its constancy when
determined by accurate methods. Our results have confirmed
the conclusions of Keyes in every way and show that the
nature and amount of the gaseous by-products formed under
uniform conditions are remarkably constant.

The same culture, or even different cultures of the same
variety, will repeat the ratio of carbon dioxide to hydrogen
with almost mathematical accuracy. This is illustrated by

2Theobald Smith, The Fermentation Tube with Special Reference to
Anaerobiosis and Gas Production among Bacteria in Wilder Quarter-Cen-
tury Book, pp. 187-232, 1893.

3F. F. Longley and W. U. C. Baton, Notes on the Determination of B.
Coli in Water in Jour. Inf. Dis., 4, 3, pp. 397-416, 1907.

4 Frederick G. Keyes, The Gas Production of Bacillus Coli in Jour. Med.
Fess 21, 1, We 69-82, 1908.

80 UNIVERSITY OF WISCONSIN STUDIES

the fact that under the conditions used in our work 264
cultures from a great variety of sources gave CO./H, ratios
varying only from 1.0 to 1.1. The amount of gas, as would
be expected, was subject to greater variation, but for a certain
type of culture under definite conditions the volume of gas
could be predicted with considerable accuracy. The kind of
gases formed in the anaerobic fermentation of sugars was
limited to carbon dioxide and hydrogen with a very small
amount of a residual gas which was undoubtedly nitrogen.
The origin of this residual gas is not certain but it obviously
has no part in the main fermentation.

A total of 615 cultures has been subjected to an exact deter-
mination of the gas produced in the anaerobic fermentation
of dextrose. The results of these determinations are as-
sembled in Table I and Fig. 1, and inelude all of the cultures
with the exception of a few from water.

pias
#02
02

> aw a
" s 5 o
irs sid poi 4 Parry tr fray toon) eas earn as ear
2 2
s 3
8 a
i. 9) 08
Q
2
are
ge fe
fa 38
ae
eG
8 2
& >
= 2
3 c
a 8
<
x
rs)
Fig. 1

In making the frequency polygons shown in Fig. 1, the
liquefying and non-liquefying cultures have been separated
and the percentages calculated for the two groups separately.
The proportion of liquefying cultures was so small that it was
necessary to make the calculations in this way. While this
distinction may seem to ascribe an undue importance to the
liquefaction of gelatin it follows the usual enstom and is
probably justified.

81

CHARACTERISTICS OF THE COLON-AEROGENES GROUP

Miilanids|had-siint | aeue= SI’ Fer 6O'T |Sl'S [IGF |29°2 Jase |IG'F [es’s lens GL tie Fe LOL is eae stor, GOS |FLOL [G8 Lb jou
Oo SINE AS paler 2 T § 9 ras LG cf cs 16 To SL t c & 4 I hae nae ra rye 6g OVE 9 “‘DIT-TON
FULL |GP'L GPL {LA'S JAC°RT TLS |ZFIT |LA' JIL [See joker fo ROE JGR TL GGFR TD Poot pers GFL eBoy
aL T T ; $1 Il 8 is ; $ To fcetfecce e I ae Cee eens res Pees I Pee Ceres eee DIT
oe TL |9T* OT 08 9° |aL°S ODS |9GF j9G'L Jet's |ze'F [98° ‘OFS [GET [96° *9 ages cs (eh'G 09°6 |G" TF |S6°
él I T g las 20 pe 1g OF cs 16 Te ie 4 ° F L G ‘e 09 9G O° | apeee teers. [810.L

5 bal nN fe ie i eS I bx pe i i S ae in a a a tn S &

é = a ~ os) to at © I an i x) bo e y 3
ey Pee ae hae Re Be a ae | | ee a a ae ae La me!
So © o uN a in > oo is n 2 a) wo x a on ~ we to - 200
OLLVY *H:*00 Ad SHYALTIAO JO NOLLAAINLSIA

I aTavii

82 UNIVERSITY OF WISCONSIN STUDIES

This figure shows these 615 culturcs in three distinct groups
based on the relation of the volume of carbon dioxide and
hydrogen produced. By far the larger number produced
carbon dioxide and hydrogen in almost equal amounts, thus
giving a ratio of approximately 1:1. The average was
CO,H.=1.06, with only an occasional culture giving less
CO, than H, and comparatively few varying appreciably on
the other side of the mean. It was observed that as the
technique of the gas determinations became more refined, the
variation in the gas ratio became less, and it is probable that
the greater part of the variation shown for this group is in
the determinations rather than in the fermentation. The
volume of gas produced by this group is relatively small.
From 10 ¢. «. of 1 per cent dextrose broth about 14 ¢. ¢. of
gas was produced. Of the 350 cultures giving the 1.06 ratio
only 3 liquefied gelatin. These will be considered later with
the liquefying cultures.

Distinetly separated from this group so far as the gas
ratio is concerned is a second type characterized by the pro-
duction of considerably more CO, than H,, by a wider range
of ratio, and by a greater variation in the gas ratio of in-
dividual cultures. Nearly all of the liquefying cultures be-
long in this group. A study of the tables and figures im some
of our previous papers will show that the actual amount of
hydrogen produced is nearly constant throughout these two
groups and that the increase in the ratio is brought about
almost entirely by a larger produetion of carbon dioxide.

In our first paper’ we suggested, as a possible explanation
of the remarkable constancy of the B. coli ratio of
CO,,/H,— 1.06 and the variation found in the aerogenes ratio,
that the equal volumes of carbon dioxide and hydrogen were
produced by a fermentation common to both groups while
in the aerogenes group there occurs an additional and in-
dependent fermentation producing carbon dioxide only.

Clark has shown that while an increase of sugar in the
medium has no appreciable effect on the relation of CO, to H,

© Loc, cil,

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 83

in the gas produced by the low ratio group, a similar change’
in the composition of the medium materially affected the gas
ratio of the high ratio group by increasing the CO,. The
possibility of distinct fermentations, one producing CO,
and H, in equal parts and one producing CO, independently
of the hydrogen, is strengthened by the fermentation observed
in the third group which gives carbon dioxide only and for
which the ratio may, for convenience, be designated by the-
«sign. Only twelve cultures of this type were found, but
the unusual end product of their fermentation entitles them
to consideration as a separate type.

The statement by Burton and Rettger® that we believe that
the two reactions are mutually dependent does not express the
opinion we have held on this subject. On the contrary we
have found considerable evidence to lead us to believe that
these fermentations were independent. Burton and Rettger
have taken a similar view and have elaborated an hypothesis
to account for. the two fermentations. They assume that in
one fermentation nearly equal volumes of carbon dioxide
and hydrogen are produced with sufficient acidity to suppress
the second fermentation when the former predominates. In
the second fermentation butylene glycol is fermented with
the formation of considerable quantities of gas and acetyl-
methyl-carbinol, the substance giving the Voges-Proskauer
reaction. Since the end products of the reaction are not acid
in their character, a proteolysis is permitted and sufficient
ammonia to account for the alkaline reaction sometimes ob-
served is produced.

CORRELATION OF OrHER CHARACTERS WITH THE Gas Ratio

The ‘‘Methyl red test.’’—It has been shown by Michaelis
and Marcora’ that the growth of a colon culture ceases when
a certain hydrogen ion concentration is reached. This re-

‘lL. V. Burton and L. F. Rettger, Correlations in the Colon-Aerogenes
Group in Jour. Inf. Dis., 21, 2, pp. 162-195, 1917.

7L. Michaelis and F. Marcora, Die Stuerproduktivitit des Bacterium
Coli in Ztschr. f. Immunvititsforsch. uw. exper. Therap., Orig., 14, 2, pp.
170-173, 1912.

84 UNIVERSITY OF WISCONSIN STUDIES

lation of hydrogen ion concentration to growth has been
worked out in considerable detail for this group by Clark
and Lubs® and was made the basis of the ‘‘methyl red test.”’
The exact point at which growth stops is dependent in some
measure on the buffer effect of the medium and, therefore, is
constant only under fixed conditions. If the buffer and
fermentable constituents of the medium are properly pro-
portioned, the limiting hydrogen ion concentration is reached
and the fermentation stops. If, on the other hand, the
sugar content is low and the buffer action high, the sugar may
be entirely fermented without raising the hydrogen ion con-
centration to the point at which fermentation stops. In this
ease the reaction ‘‘reverts’’ to the alkaline side. This action
is very well illustrated by chart I of Clark and Lubs’ paper.
It is pointed out, in this paper, that this ‘‘reversion’’ cannot
be due to the production of ammonia as has been commonly
assumed.

Unpublished work of Ayers and Rupp of this laboratory
has shown that this phenomenon is brought about by two simul-
taneous, but independent, fermentations. While the sugar
is being fermented, a secondary fermentation is converting
the salts of the acids, which are the end products of the first
fermentation, to gases, carbonates and other products.

The reaction at any given time is dependent on the relative
rates at which these two fermentations are progressing.

The change from an acid reaction toward the alkaline side
is not due, as has been assumed, to a neutralization by ammonia
from a protein decomposition but to an actual decrease in
the acids through a secondary fermentation and in some
measure to a neutralization by carbonates which appear as
end products of the decomposition of the acid salts.

The work of Harden,” Harden and Walpole,?° and Thomp-

* Loe cit,

’ Arthur Harden, The Chemical Action of Bacillus Coli Communis and
Similar Organisms on Carbohydrates and Allied Compounds in Jour. Chem.
Soc, (London, 1901), 79, pt. 1, pp. 610-628.

1” A. Harden and G. 8S, Walpole, Chemical Action of Bacillus Lactis Aero-
genes (Escherich) on Glucose and Mannitol; Production of 2: 3-Butylene-

glycol and Acetylmethylcarbinol in Proc, Roy. Soc. (London, 1906), Ser.
3, 77, pp. 399-405. ’

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 85

son™ shows that the acids formed by the accepted types of
B. coli, and B. aerogenes are identical but that the amounts
of these acids produced by B. coli are less than those pro-
duced by B. aerogenes. The results obtained by Ayers and
Rupp show that the final difference is due primarily to dif-
ferences in the rates of the various fermentations. In the
low ratio type the acid formation proceeds faster than the
fermentation of the acid salts and a hydrogen ion concentra-
tion which inhibits growth may be reached. In the high ratio
group the fermentation of the acids is more rapid and the
initial acid reaction may be converted into an alkaline one.
With these facts before us it is easier to explain the more
complicated and more variable gas evolution of the high ratio
group.

By carefully adjusting the amount of sugar and the buffer
action, Clark and Lubs?? devised a medium in which the low
ratio cultures reached the limiting hydrogen ion concentra-
tion while the high ratio cultures fermented the greater part
of the sugar without reaching this point, thus permitting a
reversion of the reaction toward the alkaline side. The Py
value of 5.0 — 4.8 (Sorensen’s scale), reached very uniformly
by the low ratio group, gives a red color with methyl red
while the high ratio cultures are uniformly yellow. Nearly
all of our cultures have been subjected to this test and it has
been found to agree with the gas determination in every case.
With only a few cultures was there any question as to the
reaction and in these cases it was found that there was some
abnormality in the gas ratio which would justify classing
these cultures as atypical. Other workers who have used
this test have also found it a reliable method of distinguishing
between the B. coli and B. acrogencs type, but some, who have
considered it less reliable than the Voges-Proskauer test,
have apparently failed to realize that it was designed to in-

11 James Thompson, The Chemical Action of Bacillus Cloacae (Jordan)
on Glucose and Mannitol in Proc. Roy. Soc. (London, 1912), Ser. B, 84,
pp. 500-504.

"122°.W. M, Clark and H. A. Lubs, Improved Chemical Methods for Differ-
entiating Bacteria of the Coli-Aerogenes Family in Jour. Biol. Chem.,
30, 2, pp. 209-234, 1917.

86 UNIVERSITY OF WISCONSIN STUDIUS

dicate the nature of the fermentation which would take place
under certain very definite conditions. A variation in the
medium, even in the kind of pepton, may change the buffer
action so much that the test no longer serves the purpose for
which it was designed. For this reason, Clark and Lubs,¥
in a second paper, have proposed a synthetic medium which
will obviate this difficulty.

Properly speaking the methyl red test cannot be considered
as a character correlated with the gas ratio. By indicating
the group to which the culture belongs, the necessity for de-
termining the gas ratio is obviated. The chemical changes
producing the reactions on which the test depends are the
changes which produce the distinet gas ratio and the two
are thus merely indicators of a fundamental difference in the
course of the fermentation. The methyl] red test is so designed
that if it is properly manipulated it must be correlated with
the gas ratio.

The Voges-Proskauer test.—MacConkey and Clemesha
considered a positive Voges-Proskauer test as one of the
characters which distinguished B. aerogenes from B. coli. This
view was confirmed by Levine,* and Johnson and Levine,”
observed that there was a very high negative correlation be-
tween the methyl red test and the Voges-Proskauer test. In
our own work we did not use the Voges-Proskauer test at first
but after the appearance of Levine's paper, 374 cultures
which were then available were subjected to this test. The
results, which have been reported by Clark and Lubs,’® show
a perfeet correlation between the gas ratio, the methyl red~
test and the Voges-Proskauer test. In some cases, however,
the reaction was so faint and disappeared so quickly that
without careful observation it would have been overlooked.

The formation of acetyl-anethyl-earbinol is not directly de-

+ Loe. Cit,

“Max, Levine, Preliminary Note on the Classification of Some Lactose
Iermenting Bacteria in Jour, Bact., 1, 6, pp. 619-621, 1916.

“B. R. Johnson and Max. Levine, Characteristics of Coli-like Microor-
yanisms from the Soil in Jour, Bact., 2, 4, pp. 379-401, 1917.

“W. M. Clark and H. A. Lubs, Improved Chemical Methods for Differ-
entiating Bacteria of the Coli-Aerogenes Family in Journal Biol, Chem,
30, 2, pp. 209-224, 1917.

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 87

pendent on the fermentation which produces the gas and it
is, therefore, additional and important evidence of the validity
of the distinction between the high and low ratio groups.

Indol.—The formation of indol has always been associ-
ated more closely with the fceal or B. coli type than with B.
aeroyenes. The correctness of this assumption is indicated
by the results shown in Table II and Fig. 2. Over 90 per
cent of the low ratio cultures formed indol against 26 per
cent for the high ratio cultures. This characteristic is evi-
dently subject to some variation and the percentage of posi-
tive results obtained depends in some measure on the technique
adopted.

TABLE IL

PHYSIOLOGICAL REACTION OF NON-LIQUEFYING CULTURES,
ALL SOURCES

CO Low Ratio High Ratio
Ilz Number | Per cent Number Per cent
i

INO! wacsss tee eaeite Gaaead Ga + 372 92.30 72 26.66
= 31 7.69 198 73.383

Saccharose ..............-. + 157 39.05 248 91.85
> 245 FAY. 94 22 3.14

Raffinose .........6-2200 00s + 173 43.14 \ 246 90.77
_ 228 36.85 25 9.22

SEACH: .ceseisscreisis seccasseaaieiaines + 24 6.16 133 50.57
_ 365 93.83 130 49.42

TONER desler cayenne Ee + 1 .29 16 7.30
_ B42 99.70 189 92.19

Mannitol] .......-.-.0+-.46+ + aT 99.00 171 64.77
— 4 99 93 35.22

GI GOTO] cee nadie esses + o2t 93.58 140 68.63
_ oy 6.41 64 31.37

SACI: |. 24. disor eaty + 197 74.06 255 95.86
_— 6) 25.93 11 4.13

DileitOl, se snciotsionye sete + 205 51.25 86 Shs
— 195 48.75 185 68.26

Adonitol .......-..0eee ee ree + 53 15.45 137 49.28
— oo) $4.54 141 50.71

A perfect correlation with the more basic characters could
not be expected but so far as the evidence goes it forms
another distinction between the low and high ratio group.

88 UNIVERSITY OF WISCONSIN STUDIES

The fermentation of carbohydrates and alcohols—In con-
sidering the ability of these cultures to utilize different sources
of carbon some consideration must be given to the difficulty
sometimes encountered in determining if a fermentation has
actually taken place. With the ordinary culture and most
of the sugars and alcohols there is no difficulty in determining
fermentation either by the gas formation, the change in
titratable acidity, or in hydrogen ion concentration. With
some cultures the reaction is obscured by a weak evolution of
gas or a rapid reversion of the reaction.

Gelotin - $82 =106 and P- Gelatin - 524215 -3.0 Vand Pr
400 80 60 40 20 +#[- 20 40 60 80 00 100 80 60 40 20 +|- 20 40 60 80 100%
Indol. es
Saccharose,
Roffinose.
Starch.
Inutin. Leal bd ree eee eee Bee [eee (|
Mannitol. pats acti ee een! Serer Palle
Glycerol, a [ka ‘ae ee eed peed ee ee
Salicin. bi iS -
Dutcitol. erated
Adonito/. vend cnet tarebfeciedl feos Eee) eee ee | a
L N
Fig. 2

In most of the sugars the change in reaction is rapid and
distinct but in some of the alcohols, notably glycerol, the
fermentation is so slow that it may be in doubt for many
days. We have considered an evolution of gas in the absence
of any apparent change in acidity or an appreciable change
in acidity without evident gas formation as fermentations.
Figure 2 shows that while there is no perfect correlation,
there is a distinct difference in the fermentative ability of the
two groups. The cultures of the high ratio group almost
without exception ferment saccharose and raffinose while only
about 40 per cent of the low ratio group utilize these sugars.
Starch is also utilized much more commonly by the high ratio
cultures. On the other hand, more of the low ratio cultures
ferment the aleohols mannitol, glycerol and duleitol than is
the case with the high ratio group. This does not hold for
adonitol which seems to be more available for the high ratio
type. he percentage of cultures fermenting the glucoside

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 89

salicin is slightly higher for the high ratio group. Only a
very few cultures of either group are able to utilize inulin.

These results agree in a general way with those reported
by Burton and Rettger’*” and Johnson and Levine,™ except
that the latter found a higher percentage of glycerol fer-
menters among the Voges-Proskauer plus cultures. They give
no data for mannitol. In summarizing these observations we
may say that the greater fermentative ability of the high
ratio group is shown in the volume of the gas formed, the
extent of the decomposition usually effected, and in the range
of material available for fermentation. While the correla-
tions are not perfect, they indicate an additional real differ-
ence between the two groups.

Subdivision of the low ratio or B. coli group.—It has been
customary to divide B. coli into two varieties designated as
communis and communior on the basis of the fermentation of
saccharose. This classification has been partially followed by
Kligler,+® but Levine *° has made of this V and P- group
six species, including one which is still further subdivided into
two varieties. A major division is made on the ability to
ferment saccharose, and the specific and varietal distinctions
on motility and the fermentation of salicin correlated with
dulcitol and glycerol. Burton and Rettger™* suggest three
species of which one liquefies gelatin and two, designated as
communior and communis, are distinguished by the saccha-
rose fermentation.

The data on which Levine’s separation is based are not given
and, as we did not include motility in our observations, we
are unable to apply his classification to our cultures. Without
admitting the validity of basing specific differences on the
fermentation of a single sugar we have separated the low
ratio cultures in the usual way as shown in Table III and
Fig. 3. The low ratio liquefiers, which probably agreed with

1 Loc. cit.

18 Loc. cit.

27, J. Kliger, Studies on the Classification of the Colon Group in Jour.
Inf. Dis., 15, 1, pp. 187-204, 1914.

2° Loe. cit.

21 Loe. cit.

90 UNIVERSITY OF WISCONSIN STUDIES

Burton and Rettger’s group 1V included only three cultures
and are omitted. There is no difference in the proportion of
cultures forming indol in the two groups. Raffinose is fer-
mented by nearly all of the cultures fermenting saccharose and
is fermented by only a very few of the cultures which do not
ferment saccharose, but considering the chemical relation of
these two sugars. this high correlation becomes of minor
significanee. The percentage of cultures fermenting glycerol
and salicin is slightly higher in the saccharose group but the
difference is so shght that it has no significance. On the whole,
these data furnish no sound basis for separating the low
ratio group into species.

‘TABLE IIL

OHARACTERISTICS OF LOW RATIO, GELATIN NEGATIVE CULTURES

Saccharose Saccharose
(positive) (negative)
Number Per cent Number Per eent

ENOL gasvcsnsseis epnegs cave + 145 91.19 197 90.78
= 14 8.80 20 9.21

Raf OS@ -5.c5:5.s:ocenesasanrniie + 153 96.22 29 13.30
= 6 3.77 189 86.69

SUARCH A¢c6¢-densacn mio eels + 18 12.08 20 9.21
= 131 87.91 197 90.78

TMU IIAE a seeoticesephoras aah aditkantgecnsaenied + 1 0.75 0 0.00
— 131 99.25 186 100.00

Manvitol sseswsissac aces + 158 99.37 2u4 98.16
= 1 62, 4 1.88

GIy. Cer Ol | mistavecenscusrenpane mentees + 120 90.90 176 4.62
_— 12 9.09 10 5.87

PS BTCITS aveiasssise deonanseacostesaraneacbroane + 62 80.51 109 66.87
= 15 19.48 ot 33,12

DICH OL) inintenarsasyirscens + 118 74.21 91 42,12
= 41 25.78 125 57.87

PTT asic asosnd nants oorcadicieaine + 8 6.10 49 26.48
123 93.80 186 73.51

The idea of making species or even varieties on a single
fermentation seems to us a very questionable one. A. fer-
mented sugar is merely a source of carbon and, while the fer-
mentation may be a constant reaction, it does not represent

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 1

a fundamental difference, especially if it ig not correlated
with other reactions. So far as data are available, the low
ratio or B. coli group appears to be a very definite and cir-
cumseribed entity and there is no apparent reason for
separating it into specics. Varicties on the basis of saccha-
rose fermentation might be recognized but the need for these
is not very evident.

Gelatin— SPt=106 Vand P- .
Saccharose + Saccharose —
100_80 60 40 20 +|- 20 40 60 80 1/00 100 80 60 40 20 +|- 20 40 60 80 /00

Indol,

Raffinose.

Starch

Inulin,

Mannitol,

Glycerol.

Salicin,

Dulertol. Sole Aa me, he epee es aren) Pee ep Lio Peeoaboass

Adomtol. L a glace ete)

Fig. 3

Subdivisions of the high ratio group—tThe high ratio or
aerogenes type shows more evidence of being a heterogeneous
group capable of subdivision into species than the group just
considered. Johnson and Levine,?* following the earlier ‘sug-
gestions of Levine,?* have two species, in one of which all
cultures are motile and nearly all liquefy gelatin while in
the other all are immotile and nearly all fail to liquefy gelatin.
They have also a V and P— group which otherwise resembles
the aerogenes type and which they consider as intermediate
between coli and aerogenes. Burton and Rettger™ have three
groups, one of which is a liquefying spore former, one a non-
spore forming gelatin liquefier of the cloacae type and one a
non-liqueficr of the aerogenes type. In our collection from
grains we found evidences of four high ratio groups including
one of 40 liquefying cultures and one of 90 non-liquefiers with

4 Loc. cit.
3 Loc. cit.
4 Loe. cit.

92 UNIVERSITY OF WISCONSIN STUDIES

nearly identical characters. The other two included only a
few cultures and were of doubtful validity.

The value of divisions based on the liquefaction of gelatin
may be very properly questioned. It is well known that the
ability to exerete a proteolytic enzym may be lost and Burton
and Rettger think that there may be an error in determina-
tions which do not permit an incubation period longer than
20 days. If the fact of gelatin liquefaction can be established,
there is no doubt that it indicates a distinct evolution in
methods of obtaining food supply and consequently has tax-
onomie significance. The indications are that the liquefiers
of our high ratio group have other characters in common
which separate them from the non-liquefiers and we have ac-
cordingly considered them separately.

Gelatin - SO - 15-3.0 “and P+

Aaonite + Adonite —
700_80_ ov 40 20 +|- 20 40 60 80 100 100 80 _G)I_40 20 +|- 20 40 60 80 100%

Indo!

Saccharose,

Roffinose.

Starch
tnulin

Mannitol.

Glycerol.

Salicin.

Dulcitol.

Fig. 4

While there is a considerable range in the gas ratio of the
high ratio group, the variation under different conditions and
between cultures otherwise identical is so great that this
character cannot be safely used for further subdivision. Many
groupings on the basis of substances fermented are possible.
The correlation of adonitol fermentation with a definite source
has suggested the use of this character as a possible indicator
of a line of demarcation.

The differences in the two groups formed in this way are
shown by Fig. 4, which is based on data given in Table IV.
The adonite + cultures are more active in every way than
the adonite — cultures, and are especially so in fermenting

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 93

starch and mannitol. It is entirely possible that this differ-
ence is due to the fact that nearly all of the adonite + cul-
tures were freshly isolated from feces while the adonite —
cultures came for the most part from dried grains. To ac-
count for these differences in this way, it is necessary to assume
that the fermentative abilities of aerogenes become attenuated
by exposure to unfavorable conditions. While there is some
circumstantial evidence to support this view, it is by no means
established. On the evidence of the fermentative reactions
these varieties have only a little more substantial basis than
the communis and communior of the B. coli group, but, as
will be pointed out later, they may serve a useful purpose
in indicating the probable source of the organism.

TABLE IV

CORRELATION OF ADONITOL FERMENTATION WITH OTHER PHYSIO-
LOGICAL CHARACTERS

Adonitol Adonitol
(positive) (negative)
Number Per cent Number Per cent
IMAG! stencils ss snseaseetees + 61 44.20 9 7.20
_ 17 55.79 116 92.80
Saccharose ................ + 139 100.00 104 82.53
_ 0 0.00 22 17.46
Raffinose .................. + 139 100.00 105 83.33
_ 0 0.00 21 16.66
Stareh: sjsscshaeciisicenioivens + 78 59.09 26 21.13
_ 54 40.89 97 78.86
WOUND ei tcccecsieesey oes. + 7 7.52 9 8.41
_ 86 92.47 98 91.58
Mannitol “oct ti awee eieow sa + 132 97.05 p 41 32.80
_ 4 2.94 84 67.21
Glycerol: ose ey sek secede + 76 82.60 62 58.48
_ 16 17.39 44 % 41.50
SAliCIN assis sien veges + 131 99.24 117 93.60
_ 1 75 8 6.40
DuleltOl ee ccssersa sai cn desnemare + 42 30.43 22 17.59
_- 96 69.56 103 82.40

94 UNIVERSITY OF WISCONSIN STUDIES

Tue Liguerying CULTURES

Fieure 1 shows that while there are liquefying cultures in
all of the gas ratio groups, a large percentage of them form a
mode which is quite distinct from that of the non-liquefying
high ratio cultures. In other words, the cultures with a very
high ratio are nearly all liquefiers. On account of the method
of ealeulating percentages, the relative numbers of liquefying
cultures are exaggerated. The ratio of approximately 1.06 was
given by only three hquefying cultures, a number so small
that they have not been considered at this time.

502-00 £92 » 1§~30
100_80 60 40 20 +|~ 20 40 60 80 /00 100 80 60 40 20 +|- 20 40 60 80 K
T

Indol.

Saccharose|

Roffinose. |___ oF mse

Lactose,

Starch,

Inulin,

Mannitol.

Glycerol.

Saheim, Laila) wef

Dute:tol,

Adonitol. | By Peres ee

Fig. 5

An additional mode over the infinity ratio marks a phys-
iclogically distinet group. A comparison of Fig. 5 with Fig.
2 shows that in fermentation reactions, the high ratio liquefy-
ine cultures do not differ in a very marked degree from the
high ratio non-liquefiers. Indol, saccharose, raffinose, inulin,
elycerol, salicin and dulcitol are fermented in about the same
way by the two groups. Starch is fermented more actively
hy the non-liquefiers, and mannitol by the liquefiers. Tf, how-
ever, we necept the arrangement shown in Fig. + we find that
the liquefiers do not agree with either the adonitol + or adoni-
tol — types. The liquefiers differ decidedly from the adonitol
+ type in indol formation, and in the fermentation of adoni-
tol and stareh, and from the adomtol— type in the fermenta-

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 95

tion of mannitol. The lactose fermcnting lqucfiers have always
been grouped together as B. cloacue, and we are inclined to
favor the retention of this arrangemeut until more conclusive
evidence can be produced to support a revision.

Although we have only a small number of cultures which
produce only CO, in the anaerobic fermentation of dextrose,
this character indicates such a fundamental physiological de-
parture from the type that we have had no hesitation in
putting them in a class by themselves.

Active liquefaction of gelatin and the fact that 8 of the 12
cultures included in this group failed to ferment lactose sug-
gests the identity of this type with the proteus group. The
three striking characteristics of the proteus group are the
failure to ferment lactose, the liquefaction of gelatin and the
formation of characteristic swarming colonics in gelatin.
Two-thirds of our cultures agree with the first character and
all with the second. he typical colony of proteus on gelatin,
according to the original description by Hauser,’ has second-
ary colonies which appear in the medium around the original
colony. Evidently many cultures are classed as proteus which
are not known to form this peeuliar type of colony. Herter
and Ten Broeck?* mention one culture which did not form
swarming colonics until it had been passed rapidly through
a series of milk tubes and then plated on dextrose gelatin.

We studied the colony fermentation of twenty-five liquefy-
ing eultures on 5 per cent gelatin at 20°C. There was a great
variation in the type of colony, and even in different colonies
by the same culture on a single plate. Only a few of these
twenty-five cultures formed what could be described as typical
proteus colonies. In most cases the colony was round, with
asmooth margin and without out-growths of any kind. Lique-
faction appeared slowly, and the margin of the colony would
be unliquefied. There were many exceptions to this, some of
which are shown in Fig. 6. At @ is shown a single strand

23 Gustav. Hauser, (ber Féulnissbacterien u. deren Beziehungen cur Sep-
ticemie, Leipzig, 1885.

2 C, A. Herter and C. Ten Broeck, A Biochemical Study of Proteus Vul-
saris Hauser in Jour. Biol. Chem., 9, 6, pp. 491-511, 1911.

96 UNIVERSITY OF WISCONSIN STUDIES

more highly magnified than the other figures in the cut. The
plate became filled with secondary colonies of this kind con.
nected by almost microscopic filaments. At d is another type
in which the colony is almost entirely filament with an oc.
casional bud-like colony. The filament formation is fre.
quently limited to fringe-like outgrowths on the margin, as,

Fig. 6

shown by the sections of liquefying colonies at ¢ and f. Some-
times on a plate filled with smooth, round, solid colonies, an
occasional colony like b or gy may develop. The type of colony
from an individual culture may be variable and, on a
single plate, may be found solid smooth colonies, large colonies
liquefying in the center, and almost any one of the colonies
shown in Fig. 6. Sub-cultures have been made from the dif-
ferent types of colonies of a single culture, but these sub-

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 97

cultures were identical in all their reactions. So far as the
evidence obtained from these twenty-five cultures goes, there
1s no relation between the type of colony and the fermenta-
tion of lactose or the gas ratio. It seems that the tendency to
‘form outgrowths is an expression of an attempt to push out
into the medium where conditions are more favorable. Some
strains have developed this tendency to a greater extent than
others, but it is very evident that it could not be placed on a
par with physiological characters for taxonomic purposes.

The infinity group liquefies gelatin more actively than the
cloacae group, but its fermentative ability, as shown in Fig.
5 is less. A comparison based on only twelve cultures must
be tentative, but some of the differences are of special interest.
This is the only group in which the correlation between sac-
charose and raffinose is not nearly perfect.

The amount of gas formed from dextrose by the infinity
group is usually much less even than that obtained from
the low ratio cultures. Traces of gas and a slight change
in the hydrogen ion concentration sometimes observed in
lactose broth indicate that there may be a feeble fermentation
of this sugar by those cultures classed as lactose negative.

The general characters of the cultures of the infinity group
identify them with the proteus type. While a few of them
differ from the usual conception of protcus by fermenting
lactose, it seems to us that the nature of the fermentation as
indicated by the end products is so much more basic than the
nature of the material fermented that the separation into
species should be made on the gas ratio rather than on the
failure to ferment lactose. .A number of cultures sent to us
from other laboratories as proteus failed to ferment lactose
but gave the carbon dioxide-hydrogen ratio of the aerogenes
group. It has been customary to include in the colon-aero-
genes group only cultures that ferment lactose. This is
largely a matter of convenience and there is no good reason
why dextrose + lactose — ecnltures should not be included.

7

28 UNIVERSITY OF WISCONSIN STUDIES

THE DIsrriBsuTION OF THE COLON-AEROGENES GROUP

When physiological or morphological characters are cor-
related with a definite habitat, their taxonomie significance is
greatly increased. The restriction of species of the higher
animals to a more or less definite habitat is too well known
to need discussion. Among the plants this relation is even
more marked and the fungi are sometimes limited in their
habitat to a single species of host plants. The source of the
various tentative groups into which we have divided our 689
cultures should be in a way a test of the validity of this
grouping. The distribution of these groups according to their
origin is shown in Table V and Figure 7.

TABLE V
SHOWING THE DISTRIBUTION OF CULTURES ACCORDING TO ORIGIN

Human Bovine Water Milk Grains
Type Feces i Feces
No.|Per cent||No.)Per cent||No.;Per cent||No.|Per cent||No.|Per cent
CO:
—--- 106 Sacech. + .......... 23 14.45 86 54.08 27 16.98 21 13.20 2 1.25
He
COs
—--,1.06 Saech, — .......... & 38.07 61 27.98 32} 14.67 37 | 16.97 5 2.99
He
CO2
—--- 1.5-3.0 Adonite + ....) 46} 33.09 1 71 46 | 33.09 32 | 23.02 14 | 10.07
He
i
Cis
-—- 1.5-3.0 Adonite — ....| 0 ]......... OF ba as a segs 14 13.59 12 «(11.65 77 | 74.
COz
CO:
Gel. liquef. aati SS na Gee cak 10) leas ce veers Of sag neat oh 4 83.35 1 8.33 7} 58.83
He |
COz | a
Gel. liquef. —--- 1.53.0 ...... 0, lnurnames He Oo ase ian 9 15.51 11 18.96 85; 65.51
He |

This collection eannot be taken as accurately representing
the relative distribution, because in some eases the isolation was
selective, as, for instanee, when a special attempt was made
to get high ratio cultures from feces. No attempt was made
to isolate liquefiers and the results may be misleading in that

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 99

they show no liquefiers in feces where they no doubt oveur
in small numbers. Water aud milk can hardly be considered
as the habitat of bacteria of this kind. They occur and multi-
ply there, but these fluids ust be looked upon as carriers
into which the bacteria have been intrcduced from some other
source. This statement will probably hold also for erains
which may be merely carrying soil or fecal organisms
mechanically.

Grains

coz
“aa 7106, Saccharose,+

Soa =106, Saccharose.—

$92 =15-30 Adonitol. +

$04 =/5-30 Adonitol—
Gel. liquel $Pz=00

Gel. iquef $92=15-30

the most striking feature brought out by Fig. 7 is the
very great preponderance of the low ratio groups in feces,
and especially in bovine feces from which, in spite of the fatt
that a special effort was made to isolate high ratio cultures,
only a single one was obtained in 150 cultures isolated. They
were more frequent in the human feces, although B. coli was
in great preponderance there also. There was great variation
in individuals and it is difficult to say what the proportion of
LB. coli to B. aerogenes would be, but it would probably vary
from 10 to 1 to 100 to 1.

This seems to agree with the earlier work which touches
on this point. MacConkey,?’ in 241 cultures from human
feces, found none that were V and P+. Ferreira, Horta and
Parades?* found 8 V and P+ cultures in 117 from human
feces. Clemesha”® says that aerogenes (V and P-++) cultures
oceur very rarely in feces. This all confirms our conclusions

2 Alfred MacConkey, Lactose-fermenting Bacteria in Feces in Jour.
Hyg., 5 3) pe. 333-379, 1905,

2A. Ferreira, A. Horta and C. Parades, Recherches sur le B. coli com-
munis de Vintestin de Vhomme in Archiv. du Real Inst. Bact. Camara
Pestana, 2, fac. 2, pp. 153-197, 1908.

22°W. W. Clemesha, The Bacteriology of Surface Waters in the Tropics,
London, 1912.

100 UNIVERSITY OF WISCONSIN STUDIES

that the predominant organism of the intestinal tract is the
low ratio or B. coli type.

Figure 7 indicates that the saccharose fermenting type is
more common in bovine than in human feces. It is not safe,
however, to put too much weight on this deduction. If we
were to assume that the failure to ferment saccharose in-
dicated human origin, we would be obliged to conclude that a
majority of the B. coli cultures from milk came from human
feces. Unequal rates of multiplication by’ the different types
may completely rearrange the original relations.

The origin of the cultures of the two groups into which non-
liquetying high ratio cultures have been divided is perhaps of
some significance. Of the 139 adonitol+ cultures, 33 per
cent were from human feces, 33 per cent from water, 23 per
cent from milk and only 10 per cent from grains. On the
other hand, 74 per cent of the adonitol— cultures were from
grains and none at all from feces. In other words, all of the
high ratio non-liquefying cultures from feces fermented
adonitol while nearly all of those from grains failed to fer-
ment this alcohol. If we assume that the 14 adonitol + cultures
found on grains came originally from feces, we have a sharp
distinction between the fecal and non-fecal types. In water
and milk the fecal tvpe predominates. It is not surprising
that this should be so since the greater part of the colon-
aerogenes cultures isolated from waters would naturally come
from samples more or less infected with fecal matter. It
may seem peculiar that so large a percentage of the fecal type
occurred in milk while they were very rare in bovine feces, but
this anomaly may perhaps be accounted for by the difference
in rates of growth of different types. Mr. Ayers has observed
that while high ratio cultures will not be found in bovine
feces by the usual methods of plating, milk infeeted with
this material and incubated at a low temperature for twenty-
four hours may contain laree numbers of this type.

It will, no doubt, be suggested that the adonitol— eultures
are merely feeal cultures which have become attenuated
through existence under the unfavorable conditions found in
the soil, on the surface of grains, or in water. We have no

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 101

evidence supporting or controverting this theory. If the
loss of ability to ferment adonitol is effected only after many
generations under the new conditions, it is only another way
of saying that a variety has been created by a change of en-
vironment. If the change takes place in the original cells,
it may be considered as an indication of the remoteness of the
contamination.

While none of the liquefying cultures in this collection
were isolated from feces there is no doubt that they occur
there under certain circumstances. Ford*®® reports the oc-
currence of B. cloacae in feces. Clemesha* says that B. clo-
acae may be present in human feces in numbers as high as
15 or 20 per cent of the total. MacConkey* found occasional
gas forming liquefiers in feces. On the other hand, nearly
one half of the coli-like organisms isolated from soil by John-
son and Levine*®* were liquefiers. Unless we assume that the
liquefiers of this group are more resistant to unfavorable con-
ditions than the other types and therefore persist longer in
water and in the soil it is evident that the principal source
of B. cloacae and B. proteus is outside of the intestinal tract.

CoNCLUSIONS

It may be considered as established by the results reported
here, supported as they are by the observations of many
other investigators, that B. coli (low ratio) and B. aerogenes
(high ratio) are very distinct types. This is based on: (1)
a fundamental difference in the course of the fermentation.
Carbohydrates are fermented more readily by B. aerogenes
and B. cloacae with a secondary rapid fermentation of the
by-product, resulting in a greater production of CO, and a
decrease in acidity.. The fermentation brought about by

%°W. W. Ford, Classification of Intestinal Bacteria in Jour. Med. Res.,
6, 1, pp. 211-219, 1901.

"1°W. W. Clemesha, The Bacteriology of Surface Waters in the Tropics,
London, 1912.

32 Alfred MacConkey, Further Observations on the Differentiation of Lac-
tose-fermenting Bacilli with Special Reference to those of Intestinal Origin
in Jour. Hyg., 9, 1, pp. 86-103, 1909.

%3 Loc. cit.

102 UNIVERSITY OF WISCONSIN STUDIES

B. coli is of a similar nature, but the primary and secondary
fermentations proceed at different rates and the end products
are different from those produced by B. acrogenes. (2) With
this are correlated various other characters, particularly a
higher percentage of indol formers in B. coli and the forma-
tion of acetyl-methyl-carbinol by B. aerogenes. (3) There is
also correlated with these physiological differences a difference
in habitat. B. coli is the characteristic gas-forming organism
cf animal feces and is commonly found elsewhere only in
localities recently contaminated with fecal matter. B. aero-
genes and B. cloacae, on the other hand, occur rarely in bovine
feces and in relatively small numbers in human feces, but are
common in soil and materials contaminated with soil.

B. coli is probably of specifie rank and the subdivisions
usually made on the basis of saecharose fermentation should
not be looked upon as more than varieties of doubtful validity.
It is possible to make two similar varieties of B. aerogencs on
the basis of the fermentation of adoritol. This character is
givcn an added significance by a high correlation with habitat.
All of the aerogenes cultures from feces fermented adonitol
while a large percentage of those from grains were adonitol
negative.

The distinction between B. acrogceues and B. cloacae, beyond
the liquefaction of gelatin, is not very sharp, but everything
considered B. cloacne should be a separate species.

A small group of cultures distinguished by the liquefaction
of gelatin, the fermentation of dextrose with the formation
of CO, only, and the fermentation of saccharose but usual
failure to ferment lactose is evidently identical with B. pro-
teus. The failure to ferment lactose, usually considered
the distinguishing character of B. proteus, does not coincide
perfectly with the more fundamental character indicated by
the single gascous end product of the fermentation. The
unusual colony formation commonly looked upon as peculiar
to proteus is probably shared with some B. cloacae cultures.
A more logical separation between B. proteus and B. cloacae
would be on the end products of the fermentation rather than
on the nature of the material fermented.

CHARACTERISTICS OF THE COLON-AEROGENES GROUP 103

B. cloacae may be considered as the stem from which the
other members of the group have sprung. By its vigorous
habits of growth, the wide range of substances from which
it is able to obtain its supply of carbon and its ability to ex-
crete proteolytic enzymes, it is able.to thrive under the sapro-
phytic conditions in which it is found. B. aerogenes has lost
the proteolytic ability and acquired semi-parasitic habits.
B. coli is still more parasitic in its habits, is more restricted
in its range of carbon supply, and is further removed from
B. cloacae and B. aerogenes by the loss of the carbinol reaction
and by the development of the ability to form indol. Still
more parasitic in their habits and correspondingly more re-
moved from B. cloacae by their physiological characters are
B. enteritides, B. typhosus and B. dysenteriae. Removed
from B. cloacae in the opposite direction is B. proteus which
has developed the proteolytic ability but lost in the utilization
of carbohydrates both in the range of materials fermented and
in the extent of the fermentation.

THE IDENTITY OF AMERICAN AND FRENCH
SPOROTRICHOSIS*

Davin JoHN Davis

INTRODUCTION

The reason for presenting this subject at this time will
no doubt be deemed adequate by those who have followed the
literature on sporotrichosis during the past several years.
This disease is known to be relatively common in France,
the number of cases observed now running into the hundreds.
In America the disease is being commonly reported in both
man and horses; the number of human cases now closely ap-
proximates a hundred, and several extensive outbreaks in
horses in different localities have been observed.

The disease is known under the name of Sporotrichosis in
both countries. In France and generally on the continent,
also in certain other parts of the world, the cause is given as
the Sporotrichum beurmanni. In the United States the causal
organism is generally but not uniformly recognized as Sporo-
thrir schenckii. Certain writers here, now and then, refer
to the organism from American cases as Sporotrichum beur-
manni or as Sporotrichum schenckii-beurmanni. The impres-
sion is general on the continent and especially in France that
the American and French organisms are distinct and that
we have to do with two different though closely related dis-
eases. It is my purpose in this paper to analyse the existing
data and to present certain new data bearing upon this matter
of the identity or non-identity of these two infections.

This discussion does not concern other distinct varieties
of Sporotricha either pathogenic or non-pathogenic. The ex-
istence of these is recognized. Many saprophytic sporotricha

* rom the Department of Pathology and Bacteriology, University of
Illinois ; College of Medicine, Chicago.

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 105

grow in the soil, in water, and under other conditions; certain
varieties are pathogenic for plants.

Infection with varieties pathogenic for man, excepting the
two here under consideration, are apparently very rare. The
following are mentioned in this connection.

Sporotrichum dori, an organism isolated from a human case
and very imperfectly described by Dor in 1906, was evidently
a different organism morphologically, culturally, and in its
pathogenicity for animals. The culture has been lost and
rio similar organism has since been found.

Sporotrichum indicum, an organism described by Castellani
in 1908, was isolated from two cases of sporotrichosis in Cey-
lon. It is impossible now to compare them with other varie-
ties since these cultures have also been lost. From the orig-
inal description given by Castellani it is clear that the or-
ganisms are very similar to, if not identical with, the French
and American varieties. He says that ‘‘it closely resembles
Sporotrichum beurmanni; the mycelial threads are somewhat
larger, between 2 and 3 microns wide; spores roundish (3
to 5 microns in diameter) or oval (4 to 5 microns long and 3
to 4 microns in breadth). Colonies on maltose agar may be
of various colors,—greyish, light brownish, dark brownish,
black’’.t There are no differential characteristics here that are
important and I am inclined to believe these strains are identi-
cal with the American variety of Schenck. De Beurmann
and Gougerot provisionally classify it as Sporotrichum beur-
manni var. indicum.

Sporotrichum gougeroti is an organism isolated from a case
in France by Gougerot who thought it different from the
Beurmann type in several respects, chiefly in macroscopic
growth and pigment production. De Beurmann and Goug-
erot observed one case only.

The Sporotrichum jeanselmei was isolated by Jeanselme and
Chevallier in 1910 from a human case and a second case
appeared as an experimental infection accidentally obtained
in the laboratory from the culture of the first case. According
to de Beurmann and Gougerot this organism is very similar

1Castellani & Chalmers, Manual of Tropical Medicine, London, 1910.

106 UNIVERSITY OF WISCONSIN STUDIES

to Sporotrichum beurmanni, deed identical with certain pleo-
morphic forms of the latter. They make the significant state-
ment in this connection: ‘‘Plusicurs de ses pléomorphismes
s‘identifient au Sporotrichum Sehenki et au Sporotrichum
Beurmanni, voire méme au Sporotrichum Gougeroti. Cette
unification dans les pléomorphismes est la meilleure preuve
dune commune origine de ces germes.’’ Their position seems
to be that this organism, the American organism Sporo-
trichum = schenchii, and the Sporotrichum beurmanni were
originally one and the same organism, and certain strains
later developed pleomorphic characteristics leading to new
varieties.

A new pathogenie sporotrichum, Sporotrichum councilinani,
described very recently by Wolbach, Sisson and Meier? of
Boston was found in a case of acute arthritis of the knee
fellowing injury. From their deseription it appears quite
different from all other strains of Sporotricha. They
summarize the distinguishing features as follows: ‘‘(1)
its pleomorphic growth, characterized by a free aerial growth
of hyphae; (2) the abundant spore formation, large size of
the spores and absence of lateral spore clusters, and (3) the
occurrence in lesions as septate, branching filaments.’’ The
last character is especially significant. The clinical history
and the character of the lesion in the patient are also of in-
terest and are possibly of importance for differential purposes.

For the purpose of this paper [ think we may eliminate
Sporotrichum dori as being quite diffarent from the other
sporotr:vha; also Sporotrichum councitinant, The very rare
cases of so called Sporotrichum indicum, Sporotrichum jean-
selmet and Sporotrichum gougeroti ave much more closely re-
lated to Sporotrichum beurmanni, the first two indeed being
probably identical with it. The existence of these very rare
varicties is here recognized but this fact is only indirectly re-
lated to the main question at issue here, namely, the possible
identity of the organisms causing sporotrichosis as commonly
observed in America and in France.

In the further analysis of this question it will be desirable

2 Journal of Medical Research, 36, p. 837, 1917.

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 107

to discuss briefly the history of sporotrichosis in America and
France and also in the other countries where it has been ob-
served.

SPOROTRICHOSIS IN NortTH AMERICA $

The recorded history of this disease in America is brief
and simple. In 1898 Schenck® reported a case of chronic
subcutaneous abscesses from which he isolated in pure culture
a fungus which grew readily on artificial media and which
was identified by Dr. Erwin F. Smith of the U. 8. Dept. of
Agriculture as belonging to the genus Sporotricha. The or-
ganism was found to be distinctly pathogenie for mice and
dogs and from the characteristic lesions the same fungus was
recovered pure. Thus, in the first case observed, all of Koch’s
laws were fulfilled. Illustrations of the human lesions, cul-
tures, and microscopic appearance of the fungus accompany
the paper of Schenck.

In 1900 Hektoen and Perkins‘ observed and very carefully
described a second case in which the fungus was isolated in
pure culture and its pathogenicity for various animals deter-
mined. They were able to confirm the results of Schenck and
after a careful comparison of the fungi from both cases con-
cluded that they were identical. Schenck also examined their
strain and pronounced it identical with his organism. They
definitely named this organism Sporothrix schenckii at this
time. Therefore it is to be noted that in two of the most
prominent medical publications of the time, an accurate
clinical, pathological, bacteriological, and experimental de-
scription of this disease appeared.

A case reported in 1899 by Brayton® agreed clinically with
the case of Schenck and of Hektoen. The organism was not
detected however and cultures were not made. Definite state-
ments as to the nature of the infection cannot be made though
it was probably a case of sporotrichosis.

- Johns Hopkins Hospital Bulletin, 9, p. 286, 1898.
‘Jour. of Exp. Med., 5, p. 77, 1900.
* Indianapolis Med. Jour., 18, p. 272, 1899.

108 UNIVERSITY OF WISCONSIN STUDIES

The next report from America was that of Duque® in
Cuba in 1908.7 Duque reported three cases that clinically
were disseminated, gummatous sporotrichosis. They were
treated surgically and in two the amputation of an extremity
was resorted to without result. Under treatment of iodide
of potassium they all responded promptly and made complete
recoveries in periods of from one to two months. Details
concerning the diagnosis are not given. Duque states that
the diagnosis was made by a careful examination of the pus
but does not say whether or not cultures were made. From
the clinical and therapeutic data we should judge that these
cases were sporotrichosis, but the lack of more complete
pathological data prevents further analysis.

Burlew* in 1909 observed a case of sporotrichosis of the
disseminated gummatous type in a farm laborer in Santa
Anna, California. Both legs and the face were involved. The
organism was cultured and identified as the Sporothrix schen-
ck.

From this year (1909) to the present time the number
of cases recognized and reported has increased rapidly so that

“American Jour, Dermat. and Gen, Urin. Dis., 12, p. 240, 1908.

™The period of eight years lapsing between the reports of Hektoen and
Perkins and those of Duque, Burlew, Hyde and Davis, ete., in which no
cases appeared has been’commented upon by de Beurmann and Gougerot.
Bull. et Mem. de la Soc. Med. des Hospit. de Paris, 35, p. 798, 1910. They
infer that sporotrichosis in North America had practically been forgotten
and that only after attention had been called to this disease through the
Jater work of the French did Americans begin again to recognize this dis-
ease. There is little truth in this statement. Naturally the large amount
of work that was being done by the French between 1906 and 1908 did at-
tract attention in America. However, it is surely not correct to state that
the disease had been forgotten here when men like Hektoen, Welch, Smith
and others, who had recognized or seen the disease and the fungus, coi-
tinued to engage in active work in pathology. The real reason no doubt
was the fact that the disease in the human is restricted as we shall pres-
ently see, almost entirely to the valley of the Missouri River. This lo-
cality in the central and western portion of the country, especially at that
time, was not developed medically and naturally only the cases which
drifted out of the region to medical centers would be apt to be detected.
Such apparently was true of the cases of Schenck and of Hektoen and
Perkins. In later years when men like Sutton and others worked in that
locality cases were recognized in much larger numbers.

® South. Calif. Pract., 24, p. 1, 1909.

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 109

now they can no longer be considered rare. Ruediger’
gathered together and analysed all the cases in the United
States in 1912. He found 57 in which the diagnosis had been
made with reasonable certainty. Since then the literature
each year has furnished a considerable number of additional
reports. Many cases have no doubt not been recorded in the
literature. The writer is aware of several in which cultures
were obtained and identified as Sporothrixr schencku but never
reported.

Ruediger called attention to the interesting fact that the
disease oceurred chiefly in the Missouri River Valley. Five-
sixths of the 57 cases were from this locality,—the others
being scattered more or less diffusely over the country. North
Dakota, which furnished 22 authentic cases, seems to be the
chief focus of human infection in this country. Kansas has
also furnished a large number of cases. A map showing the
location of the cases in the United States accompanies Ruedi-
ger’s paper and brings out strikingly the geographical dis-
tribution. However, K. F. Meyer?’ has more recently analysed
the data, especially those dealing with the relation of animal
and human sporotrichosis in this country. He shows that
ag new cases appear it becomes increasingly evident that the
disease is widely distributed, though certain localities like
the Missouri River valley furnish the great majority of the
cases. It has been reported from the following states: Mis-
souri, Kansas, Iowa, Nebraska, Texas, Virginia, West Vir-
ginia, Ohio, New Jersey, District of Columbia, South Dakota,
North Dakota, California, Illinois, Pennsylvania, New York,
Minnesota, Wisconsin, Indiana, Montana and Michigan. Two
cases have been reported from Canada.

Sporotrichosis has appeared in horses in several localities
in the United States. The disease was recognized clinically
in horses some time before it was accurately studied bacter-
iologically in this animal. Horses were found in 1908 or be-
fore in North Dakota suffering from what was then taken to
be mycotie lymphangitis. From the description and the

° Jour. of Inf. Dis., 12, p. 193, 1912.
wy, A, M, A., 65, p. 579, 1915.

110 UNIVERSITY OF WISCONSIN STUDIES

illustrations given in the Second Annual Report (1908) of
the Live Stock Sanitary Board to the Governor of North
Dakota, it is evident that clinically these horses were afflicted
with sporotrichosis. Furthermore, organisms obtained from
these Dakota horses were declared to be identical by the
Bureau of Animal Industry in Washington with the organisms
from certain horses afflicted with a similar disease in Penn-
svlvania, from which an organism was isolated and clearly
shown to be the Sporothrir schencktt. The Dakota organism
was at first taken to be Succharomyces farciminosus, the cause
of lymphangitis in horses as deseribed by Tokishike and Pallin,
Through the comparative studies of Paige, Frothingham and
Paige, and also of the writer, of the organisms isolated from
the Pennsylvania horses and an organism isolated from a
human case from North Dakota by the writer, it was shown
that the organisms were without question identical. This
established the first vlear identity of the organism from lesions
in horses and in the human and showed too that it was ap-
parently identical with the Sporotrichum schenckii as
described by Schenck, Hektoen and others.

Kk. F. Meyer™ has also recently studied this disease in
horses. He concludes that spontaneous sporotrichosis in this
animal is very common, especially in two localities, Penn-
sylvania and North Dakota. He cites a ease of accidental
laboratory infection in man as proof of the pathogenicity of
equine strains for the human. The evidence collected, how-
ever, does not support the theory that sporotrichosis is very
frequently transmitted from horse to man in the United
States. His opinion is that the Sporotrichum schenckii, Sporo-
trichum beurmanni, the organisms from mules and horses in
Madagascar, and the South American strains are all identical.
He proposes the use of the term Sporotrichian schenckii-beur-
mann for all.

1 Loc. cit.

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 111

SPOROTRICHOSIS IN FRANCE

In France the history of this disease begins with the report
of a case by de Beurmann and Gougerot in 1903. Apparently
they completely overlooked the work of the American in-
vestigators published several years earlier and thinking they
had discovered a new organism they submitted it to Matruchot
and Ramond who identified it as a sporothrix and in 1905,
in a note to the Biological Society of Paris, named it Sporo-
trichum beurmannt, A second case was observed in France
in 1906 by de Beurmann and Gougerot and later they identified
other cases and made numerous extensive and admirable
studies on all phases of the disease.1?, New cases rapidly ac-
cumulated in the French literature and it was soon evident
that the disease in that country was not rare. It was observed
also in the dog and the horse, the organisms being identical
with that from the human. It is to be noted that not until
1906 did the French learn of the American cases and of the
Sporothrix schenckii. French workers generally contend that
the Sporotrichum beurmanni is different from the Sporothrix
schencku.

SPOROTRICHOSIS IN SouUTH AMERICA

In 1907 Lutz and Splendore™* in San Paulo in Brazil were
the first workers to recognize spontaneous sporotrichosis in
lower animals. They observed the disease in both gray and
white rats. They also reported five human cases from the
same locality. They noted that the disease in rats was
transmitted through bite wounds usually on the extrem-
ities or tail and following an initial lesion a generalized
infection would result. Transmission from the rat to man,
while probable, was not demonstrated. These studies, it
should be noted, were made independently, the work of Ameri-
ean and French investigators not being known to them until
some time later. The organism as described by them cor-

22 De Beurmann et Gougerot, Les Sporotrichoses, Paris, 1912.
18 Cent. fiir Bact., 45, p. 631, 1907.

112 UNIVERSITY OF WISCONSIN STUDIES

responds in detail with the North American variety and de
Beurmann and Gougerot have examined it and pronounced it
identical with the French strains. This identity has been
conceded by Lutz and Splendore.

Balino and Marco del Pont't in 1907 discovered a case of
this disease in Buenos Ayres, and Greco,’® also in 1907, one
from Uruguay. Greco suggested calling his organism Sporo-
thrichum schenckii-beurmanne. According to him it agrees
with the organisms of both Schenck and de Beurmann. Other
cases have since appeared in South America.

SPOROTRICHOSIS IN MADAGASCAR

On the island of Madagascar Carougeau’® in 1908 found
this infection in mules and in horses. It is a common disease
there. Clinically and pathologically it agrees in every way
with the disease as it appears in man. It is either a dis-
seminating or an ascending gummatous sporotrichosis and
responds promptly to potassium iodide. Carougeau repro-
duced the disease experimentally in the mule by intravenous
injection. He reports a human infection in a veterinarian
who punctured himself while operating on a sick mule. He
clearly differentiates this infection from the closely related
but more serious one of Saccharomyces farciminosus. The
sporotrichum from the mules was carefully described by
Carougeau and agrees with WSporotrichum schenckit. De
Beurmann and Gougerot have identified it with the French
organism and this identity has been acknowledged by Carou-
geau,

As to distribution, then, sporotrichosis is practically a
world-wide disease having now been noted in North America,
South America, Europe, Madagascar and probably India.
The chief focus in Europe is France but cases have been ob-
served also in Germany, Austria, Switzerland, Italy, England,
Belgium and Spain. In North America, as already stated,
it is largely confined to the Missouri River Valley.
“Argentina Med., 2, p. 23, 1908.

% Argentina Med., 45, p. 699, 1907.
“Bull. et. Mem, de la Soc. Med. de Hopit. de Paris, 84, p. 507, 1909.

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 113

Animal susceptibility is rather general, the spontaneous
disease having appeared in man, horse, mule, dog and rat. It
has been observed as an accidental infection in man. Ex-
perimentally it has been produced in a large number of the
lower animals, the rat being probably the most susceptible and
useful animal for this purpose.

A STATEMENT OF THE QUESTION

At their request, Hektoen in 1906 sent to de Beurmann and
Gougerot at Paris a culture of the American organism which
he had isolated seven years before. After studying and com-
paring this organism with their strains they declared that the
American and French strains were different and they con-
tinued to retain the name of Sporotrichum beurmanni for the
French fungus and to use the term Sporotrichum schencku
for the North American strains. It is to be noted, too, that
de Beurmann and Gougerot and their French colleagues con-
sidered the South American strains, the Madagascar strains,
the German, Austrian and other strains, all of which were
described after their work, as identical with the French
organism.

In 1910 the writer took to Gougerot in Paris a strain iso-
lated by himself from a typical human case from North Dakota
and reported later by Hyde and Davis.*7 I received from
him and also from Sabouraud at that time strains isolated
from cases in France and called by them Sporotrichum beur-
manu. I also obtained from Hektoen a culture of his sporo-
trichum which he had preserved from his case of 1899, a
culture of which, as stated above, he had sent to de Beur-
mann and Gougerot in 1906. De Beurmann, Gougerot and I,
therefore, have French strains, American strains and the
original Schenck-Hektoen strain for comparison. In order
to simplify and limit the discussion as far as possible I will
make the following statement: first, excluding for the time
being the Schenck-Hektoen strains, we may consider all the
later strains, except Sporotrichum councilmani, isolated in

N Jour. of Cut. Dis., 28, p. 321, 1910.
8

114 UNIVERSITY OF WISCONSIN STUDIES

the United States from man and horses by numerous workers
ineluding the writer identical with each other. My own work
as well as the work of K. F. Meyer, Sutton, Ruediger, Page,
Frothingham, Paige, and others, all tend to confirm this point.
The strains have usually been designated as Sporotrichum
schencki. Again excluding Sp. dori, Sp. jeauselmet and Sp.
gougeroti in France, all the French strains of sporothricha are
admitted by all, both French and Americans, to be alike.
They have been designated Sporotrichum beurmanmi by the
French. Gougerot in a publication’® made after examining
the American cultures which I gave him in 1910 admits that
they are identical with the French strains but different from
the old Schenck-Hektoen cultures. In another article on this
subject he writes as follows: ‘‘Hyde & Davis bezeichnen also
Sp. Schencki einen Parasiten welchen uns Davis ubermittelt
hat und der zweifellos identisch mit dem sp. Beurmanni ist.’’
A further statement in the same paper on this point is as
follows:

Die Beantwortung der aufgeworfenen Frage lasst drei Méglich-
keiten zu:

(1) Der Stamm Hektoen-Gougerot des Sp. Schencki stellt den-
Typus des Sp. Schencki dar; das Sp. Beurmanni muss von ihm
unterschieden werden, steht ihm jedoch nahe und stammt von dem-
selben Urstamme ab wie er.

(2) Sp. Schencki und Sp. Beurmanni stammen beide von ein
und demselben Urstamme ab; sie zeigen zwar gegenwartig einige
Verschiedenheiten, sind aber durch Zwischenglieder, wie z. B.
den Stamm von Hyde & Davis, miteinander verbunden.

(3) Der Stamm Hektoen-Gougerot des Sp. Schencki ist ein fest
gewordener pleomorpher Vertreter dés Sp. Schencki und von den
Stammen “Schencki initial” und “Hektoen initial” unterschieden.
Es ist jedoch unméglich, auf Grund unserer Kenntnisse von
diesem Stamm das echte Sp. Schencki zu _ beurteilen, weil
leizeres sich vollkommen vom Stamm MHektoen-Gougerot unter-
scheidet und beispielsweise durch den Stamm Hyde-Davis vertre-
ten ist. Da aber der Stamm Hyde-Davis mit dem Sp. Beurmanni
identisch ist, so mtissen das Sp. Schencki und das Sp. Beurmanni
ebenfalls identisch sein.

* Sporotrichosis Nord Americans: Bull. et Mem. de la Soc. Med. de
Hop. de Paris, 35, p. 798, 1910.

* Gougerot, Kolle & Wasserman, Handbuch der Pathogenen Microorgan-
ismen, 2 Auflage, 5, 225, 1913.

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 115

Die Frage kann nur entschieden werden durch Vergleich der
Original-kulturen von Schenck und Hektoen. Entschliesst man
sich, diese dritte Hypothese einer Unitat des Sp. Schencki und
des Sp. Beurmanni anzunehmen, so wurde trotzdem die Tatsach®
bestehen bleiben, dass dieser Parasit in zwei Typen sich darstal-
len kann; als Sp. Schencki, der seltener Typus, welcher einzig
von den amerikanischen Forschern im Jahre 1898 and 1900 be-
schrieben worden und von welchem der Stamm MHektoen-Gou-
gerot eine Abart bildet (Sp. Schencki-Beurmanni var. Schencki),
und schliesslich als Sp. Beurmanni, ein sehr verbreiteter Typus,
der zum ersten Mal von Matruchot & Ramond im Jahre 1903-
1905 und spater im Jahre 1906 von Beurmann & Gougerot
studiert worden ist (Sp. Schencki-Beurmanni var. Beurmanni.)

From the above statements it is evident that Gougerot him-
self concedes the identity of the French and the later Amer-
ican strains and I entirely agree with him on this point. I
have examined several French strains very carefully and
compared them with later American strains from man and
horses without noting any essential points of difference. Ad-
mitting then that the French strains and the later American
strains are identical, the question resolves itself into a com-
parison of the disease and organisins as observed by Schenck
and by Hektoen on the one hand and the French and later
American disease and organisms on the other. These latter,
it is to be noted, are conceded to be identical with the strains
from South America, Madagascar, and the other foci men-
tioned above. If it is shown that they are identical with
the Schenck-Hektoen strain, they should all be called Sporo-
trichum schenckit. If they are different, they would be called
Sporotrichwn beurmanim and the strains of Schenck and Hek-
toen would remain as isolated and unique organisms differing
from all other described sporotricha.

CoMPARISON OF THE Two CASES OF SPOROTRICHOSIS OBSERVED
By SCHENCK AND BY HEKTOEN AND PERKINS WITH
THE LATER AMERICAN CASES

Clinically these two cases were typical ascending gumma-
tous sporothrichosis identical in every way with the many
cases observed later by the French and by many observers

116 UNIVERSITY OF WISCONSIN STUDIES

here in the United States. No one, so far as I know, has at-
tempted to differentiate them on clinical grounds.

Therapeutically they responded promptly to potassium
iodide as did the later French and American cases.

The morbid anatomy and histology in the human were, so
far as studied, the same in the two types, the Jesions being
those of a chronic abscess. In experimental atimals the
lesions produced by the two types are identical as admitted by
Gougerot.

Bacteriologically there is greater opportunity to detect
minute or subtle differences should they be present and these
we shall consider in detail. In the cases of Schenck and of
Hektoen and Perkins, the organisms were not seen in the
human tissues or in the pus. In experimental animals the
organisms appeared as oval and elongated forms with ocea-
sional round forms. They stained with Gram. In no way
did they differ from the forms seen in tissues infected with
organisms of the French strains. I have made a special
study?° of the tissue forms in experimental animals using
strains from France and from America as well as the Schenck-
Hektoen strain. No differences could be detected between
them. When these various strains are grown in animal fluids,
blood ete., in the test tube, elongated forms similar to those
seen in the tissues are produced and here again no differences
between the various strains were noted.

The question of the virulence of the various strains may
not be of any importance in differentiation since this property
is such a variable one. However, it may be stated that the
Schenck-Hcktoen strain even after years of artificial culture
is still about as virulent for rats as are the freshly isolated
strains of the French type. According to the paper of
Schenck, these organisms were virulent for mice and dogs.
Hektoen and Perkins produced lesions in mice, dogs, rats and
guinea pigs (slight). As far as these results are comparable
with those obtained with the French and later American
strains, they agree in all essential points.

We now come to a discussion of the morphological and
cultural characteristics, both microscopic and macroscopic, of

“ Davis, Jour. Infect. Diseases, 12, p. 458, 19138.

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 117

the organisms and in doing this it begomes necessary to state
in detail the differences which the French workers have pointed
out between Sporotrichwm schenckti and Sporotrichum beur-
manint since they very largely center around these properties.
They have been stated succinctly by de Beurmann and
Gougerot in their article on North American sporotrichosis**

and in order to avoid misstatements I quote as follows:

Sporotrichum Beurmanni

Cultures difficiles, mais possi-
bles 4 38 degrés. Optimum 22 a
30 degrés: donc developpement
plus lent.

Sporotrichum Schenckii

Cultures facile & 38 degrés.
Optimum 30 4 38 degrés: donc
developpement plus rapide.

Aspect macroscopique des cultures sur gelose glycosée peptoneé

de Sabouraud (milieu d’épreuve).

Pigmentation rapide et com-

pléte. Colonies toujours trés
colorées, de teinte chocolat ou
noire.

Circonvolution 4 la facon des
circonvolutions celebrales.

Pigmentation trés lent, le plus
souvent inconstante ou absente.
Colonies peu colorées ou blanches
le plus souvent.

Sillons presque rectilignes di-
vergeants & partir d’un centre
comme des vallées du cone d’un
volcan.

Aspect microscopique des cultures sur lames séches et en gouttes

pendantes.

Filaments myceliens de 2 u de
large plus rectilignes, quelquefois
agrégés, mais surtout enchevé-
tres, non paralleles.

Spores de 3 sur 5 a 6 u, trés
nombreuses, inserées sur de
longs filaments ou 4 l’extrémité
de filaments lateraux courts ou
longs.

2 Loe. cit.

Chlamydospores.

Filaments myceliens de 2 u
de large plutét curvilignes,
onduleux, presque toujours
agrégés et paralleles en fais-
ceaux, sans enchevetrement
habituel.

Spores trés rares, souvent
méme absentes, inserées le long
et surtout a Tlextrémité de
longs filaments. Peu ou pas de
conidiophores courts lateraux.

Pas de chlamydospores con-
nues.

118 UNIVERSITY OF WISCONSIN STUDIES

Caractéres biologiques (Blanchetiere et Gougerot).

Fait fermenter la saccharose. Fait fermenter la lactose.
Ne semble pas faire ferment- Ne semble pas faire ferment-
er la lactose, ete. er la saccharose, etc.

Matruchot has stated the differences more in detail but
since all the essential points are covered by the above outline
it will not be necessary to state them again. I shall take up
these points in order and attempt to analyze them in the light
of data from both American and French sources.

First, as to the optimum temperature for growth, Schenck
states that for his organism it was between 20°C. and 387° C.
Hektoen says it would seem to be about 37° C. Growth is
much slower at 20° C. My own experiments have not con-
vinced me that there is any appreciable or constant difference
in optimum growth temperature between Sp. schencku and
Sp. beurmanni. Slight differences are often: observable be-
tween various strains of sporotricha. In growing many cul-
tures side by side, including the original Sp. schencku, growth
was most rapid and most abundant at temperatures from
28° C. to 32° C. Variation in optimum growth temperatures
is common in fungus organisms of this type. They are not
delicate in this respect and small differences should not be
unduly emphasized as differentiating features.

In the outline quoted above de Beurmann and Gougerot
have next emphasized certain points concerning the ma-
croscopie characters on special media of the cultures which
in their opinion are important in differentiation. These points
center chiefly round the fact that sporotricha generally are
especially prone to change and modify their eultural prop-
erties on artificial media, a character referred to as pleomor-
phism. This is so important and so much has been made of
cultural differences in distinguishing Np. schenckii and Sp.
bewrmanni that I must discuss it somewhat in detail.

First, the colonies may in a great variety of wavs alter
their pigmentation, the tints changing through various shades
of brown and black; portions or all of the culture may be pure
white. These changes may or may not be permanent. I

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 119

have for some time made a study of chromogensis”’ in these
cultures and have now some pure white strains which sprang
from deeply pigmented cultures. Indeed, from the culture
I received from Gougerot of Paris, a white colony appeared
which has remained pure white and smooth, and though tested
on numerous media of the most favorable sort (carrot, potato,
Sabouraud medium), remains pure white. Passage through
a rat for six weeks did not alter it. It has now passed through
twenty-four generations without change. The black colonies
continue to produce pigment as usual. Similar alterations
have been observed in other strains. Distinct and similar
changes have been noted in the Schenck-Hektoen strain but
they are less marked. It is interesting to note Gougerot’s
statement”? in this connection: ‘‘Par exception nous avons
eu des pléomorphismes blanes qui sont trestis irreductibles ;
ils étaient associés & des pléomorphismes de surface et ces
pléomorphismes complexes donnaient un Sporotrichum Beur-
manni, identique d’aspect au Sporotrichum Schenckii.’’

A second pleomorphism relates to form of growth, smooth-
ness, wrinkling, ete. Colonies tend to lose their irregular
and corrugated surface and become smooth and leathery in
appearance. This is a common change in strains of sporo-
tricha which can be brought about, at least to some extent, in
all strains by suitable culture, especially on ill adapted media.

A third is the tendency to form on the surface growth hair-
like processes or finely pointed spines. This is seen quite
commonly and is a striking feature of many cultures of the
original Schenck-Hektoen strain.

A fourth pleomorphic change is the appearance of a
powdery growth covering part or all of the media. The color
13 variable and may range from black through brown to pure
white. This alteration is largely dependent on surface de-
posits of spores.

" It is to be emphasized that these pleomorphic changes above
noted are common. In some strains they are far more fre-
quent than in others, but probably occur in all strains at

2 Davis, J. Tif. Diss 11, pe 174, 1915.
2 Les Sporotrichoses, Paris, p. 91, 1912.

120 UNIVERSITY OF WISCONSIN STUDIES

times. They are variable but often fixed and permanent.
They are so manifold in character that cultures show a great
variety of appearances and two strains identical at first may
later through these changes become quite different in appear-
ance. De Bewmann and Gougerot state in their monograph
on page 1383 (Les Sporotrichoses) that they have observed
strains of Sporotrichum beurimanni (notably of the race
alpha), through pleomorphic change, become identical with
Sporotrichum schenckti. Others have become identical with
Sporotrichum jeanselmet ov have even simulated Sporotrichum
gouyeroti. Again certain strains have reverted to short forms
comparable to yeast or blastomycetes. My own work also con-
firms in general the above observations of de Beurmann and
Gougerot. I have noted veast-like forms in certain strains
and a great many changes in pigmentation and other morpho-
logical appearances, some of which are fluctuating, others are
apparently permanent.

In the light, then, of the above facts it seems to me that dis-
tinctions of these sporotricha based on pigmentation become
valueless because of these easy and striking fluctuations. So,
too, surface convolutions and forms simulating the cone of a
voleano are factors which change under conditions favoring
pleomorphism.

Under microscopic aspect of cultures on slides and in hang-
ing drop in their outline de Beurmann and Gougerot consider
especially spore formation. They have repeatedly stated that
in cultures of Sporotrichum schenckii the spores are rare or
even at times absent on the filaments. This is true, at least, of
certain cultures that develop little or no pigment. As a dif-
ferentiating feature, however, this point is not necessarily
significant. I have noted other strains, especially the non-
pigmented ones, which show this same characteristic. This
dearth of spores in the strain of Sporotrichum schenckii which
they examined was, I think, no doubt due to a pleomorphic
change. It is important to note the fact, which they have not
referred to in their publications, that in the original articles
of both Schenck and Hektoen and Perkins several photo-
graphs of unstained organisms show the mycelium with spores
attached to the sides and ends in great abundance. I think

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 121

these photographs are conclusive on this point and show that
no doubt a change occurred later in these cultures resulting
in a strain bearing fewer spores. Spore formation is extreme-
ly variable as de Beurmann and Gougerot admit. Further-
more in their monograph they state that the white pleo-
morphic forms—those approaching the type of Sporotrichum
schenckti—are very poor in spores, certain ones becoming
entirely devoid of them. I wish again to emphasize this point
as practical proof that changes did result in this strain from
the time it was first described and the time, some seven years
later, when it was sent to de Beurmann by Hektoen. Further-
more, de Beurmann and Gougerot write ** that Hektoen stated
in his letter when transmitting the culture that it seemed to
have lost its power of producing spores as compared with the
preceding generations. This, I believe, is definite proof of a
change which no doubt occurred on artificial media and which
these writers have used to differentiate the Sporotrichum
beurmanni from Sporotrichum schencku. Original organisms
and original descriptions should be compared for this pur-
pose, not organisms changed through growth on artificial
media.

As to chlamydospores, de Beurmann and Gougerot state
that Sporotrichum beurmanm forms them while the Sporotri-
chum schenckii does not. Matruchot also makes this state-
ment. I discussed this matter in a special paper some time
ago”’ and showed that, at least under certain conditions, the
Sporotrichum schenckii readily forms typical chlamydospores.
This was especially true on media poor in nutrient material.
K. F. Meyer?* confirmed my results in this respect noting
chlamydospore formation not only in the original Sporotri-
chum schenckii but also in the many strains of sporotricha
which he isolated from horses in the United States.

With reference to the arrangement of mycelial filaments,
it may be stated that this is a property decidedly pleomorphic,
and with the appearance of the pleomorphic alterations noted

4 Bull. et. Mem. de la Soc. Med. des Hop., Paris, 35, p. 745, 1910.
% Jour. Inf. Dis., 15, p. 4838, 1914.
* Jour. of Inf. Dis., 16; pe 399, 1915,

122 UNIVERSITY OF WISCONSIN STUDIES

above on artificial media the mycclial filaments may run in
straight or curved bundles with little entangling.

As to biological characters the fermentation reactions of
these organisms are apparently not very uniform or definite.
Gougerot says that Sporotrichum beurmanni ferments saccha-
rose but not lactose, whereas Sporotrichum schenckiu ferments
lactose, but not saccharose. He states, however, that he does
not know whether these properties are fixed for all strains
and for the pleomorphic forms. Greco noted that his strain
from South America failed to ferment lactose, saccharose and
mannite. This does not agree with the fermentation reactions
for Sporotricham bewrmanni as given by de Beurmann and
Gougerot but they nevertheless classify the South American
strains as Sporotrichum beurmanm. At the same time they
use this difference in the fermentation of lactose and saccha-
rose to differentiate Sporotrichum beurmanni from Sporo-
trichum schenckii.

Meyer and Aird?’ have made a careful study of the fer-
mentation of American strains and of Sporotrichum beur-
manni. They were not able to confirm the finding of
Blanchetiére and Gougerot that Sporotrichum schenckii fer-
mented lactose. Furthermore, they found the fermentation
of saccharose irregular. They state that ‘‘in considering
these results purely from a differential diagnostic viewpoint
it is quite evident that it cannot be used for this purpose and
the fermentation of carbohydrates is just as little a criterion
of the type of sporotrichum as is the absence of pleomorphism
and the chlamydospore formation’’. Their conclusions are so
definite and so relevant that I quote them:

The differentiation of pathogenic sporotricha into two distinct
species by means of the fermentation of carbohydrates, is impos-
sible. The reactions are not fixed and are as inconstant as the
many variations noted in the formation of chlamydospores and,
frequently, in pleomorphism. There does exist however an ap-
parent relation between the pigmentation of the sporotrichum
strains and the ability of these strains to ferment saccharose.
The alpha and beta types are the most active fermenters.

7 Jour, of Inf. Dis., 16, p. 399, 1915.

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 123

This and other evidence, which will be presented elsewhere,
make it apparent that the American sporotricha—of which we
studied thirty-five strains—have, in many respects, type char-
acters in common with Sporothrix beurmanni. In the light of
de Beurmann’s and Gougerot’s work, some of the American
strains are doubtless Sporothrix beurmanni, and it is not per-
missible to call such strains ‘‘Sporothrix schenckii’’ merely for
the sake of simplicity. The discussion of de Beurmann and
Gougerot (28) on this subject can now also in our opinion, be
satisfactorily closed, namely: that Sporothrix schenckii, Hektoen-
Gougerot strain, is an absolutely fixed type. The true Sporothrix
schenckii is represented however by all of the recently isolated
strains. Inasmuch as most of these strains are undoubtedl;
identical with Sporothrix beurmanni, the Sporothrix schenckii is
identical with the Sporothrix beurmanni.

The American strains of pathogenic sporotricha are therefore
best classified as one species. Sporothrix schenckii-beurmanni (as
suggested by Greco.)

Having now completed the discussion of the several points
of differentiation quoted above from de Beurmann and Gouge-
rot, I shall next briefly consider certain other similarities of
the French and American strains of sporotricha that deserve
mention, it seems to me, in a discussion of their possible iden-
tity. Slight and otherwise insignificant differences between
organisms may be determined often by differences in serum
reactions in varying concentrations. In the study of this
group of organisms, Widal and Abrami showed that
positive agglutination occurred in patient’s serum in dilu-
tions often of 1/400 or 1/500 or even higher. This has been
confirmed by several workers. For differential purposes they
noted that patients suffering with other mycelial infections
like actinomycosis, nocardiosos, ete., give a positive but much
lower agglutination. Gougerot and Caraven noted that the
serum of a case of hemisporosis agglutinated in dilution of
1/400. This was evidently exceptional.

The writer immunized rabbits with several strains of
sporothrix for a*period of about 8 months. The strains in-
cluded Sporotrichum schenckii obtained from Hektoen, Sporo-

Bull. et Mem. de la Soc. Med. de Hop. de Paris, 26, p. 9, 1908.

124 UNIVERSITY OF WISCONSIN STUDIES

trichum beurmanni from Gougerot of Paris, a sporotrichum
isolated by K. F. Meyer from a horse in the United States, and
a strain isolated in 1909 by the writer from typical sporotri-
chosis in a man from North Dakota. The sera of the various
rabbits were tested with the homologous organisms and also
with other strains. It was found that agglutination appeared
quite uniformily in the dilutions of serum varying from
about 1/320 to 1/640. In most instances a slightly higher
agglutination appeared in the homologous ‘sera but this was
not always the case. The several strains tested could not be
differentiated by these interagglutination tests in animals.

Similar tests were made with serum from a human case in
which agglutination with the homologous organism occurred
at 1/160. Here again the original Sp. schenckii and Sp.
beurmanne were agglutinated at approximately the same dilu-
tion, namely, 1/160. The controls were negative.

Wilder and McCullough” studied the serum from a case of
sporotrichosis of the eye. Tests for agglutinins and opsonins
in the serum of the patient against several strains of sporo-
tricha, including the infecting strain, the original Schenck-
Hektoen strain, a French strain, an equine strain from Mever,
and two other American strains from typical cases, revealed
no specific differences in the antibody content of the serum.

The reaction of complement fixation is positive in cases of
sporotrichosis but it seems less reliable than that of agglutina-
tion. The studies of Widal and Abrami and other French
workers have shown that the results are very definite but that
an infection with many other mycoses (actinomycosis, hemis-
porosis, discomycosis, ete.) will also give a positive test. A
priort, then, one would not expect this test to be useful in
differentiating strains of sporotricha. J. J. Moore*®® in our
laboratory has made such studies, finding a definite fixation
in human serum from a case of sporotrichosis using the homol-
ogous organism. He obtained similar results when antigens
made from the various other strains, including Sporotrichum
schencku, Sporotrichum beurmanni and an equine strain, were

27. A. M. A., 62, p. 1156, 1914.
% Jour. Inf. Dis., 28, p. 252, 1918.

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 125

used. He concluded that these organisms are identical so ‘far
as this test is concerned.

Bloch** in 1909 was the first to obtain a positive skin re-
action in a case of sporotrichosis though some work had been
done along this line by de Beurmann and Gougerot in 1906
without definite results. Bloch used a ‘‘sporotrichosine’’ ex-
tracted from a broth culture. The reaction was very definite.
French workers, especially de Beurmann, and Gougerot and
Chopin, about the same time took up this work, using in-
tracutaneous injections of extracts of the killed organisms in
salt solution and obtained positive results. The reaction,
however, was shown by them not to be absolutely specific.
Other mycoses (actinomycosis, oosporosis, saccharomycosis, ex-
ascosis, at times tuberculosis) responded so that the method
was useful according to them only for the differentiation of a
rather large group. From their data, one would conclude
that this method could have no value for differentiating
closely related organisms belonging to the genus sporotricha.

Recently Moore and the writer tested a human case of
typical sporotrichosis with a sporotrichosine consisting of
killed sporothrix in salt solution. The patient was tested both
when receiving and not receiving potassium iodide. A very
distinct skin reaction was obtained with the sporotrichosine
made from the original Sporotrichum schencku and also from
a strain of Sporotrichum beurmanni obtained from Gougerot.
No differences were noted in these reactions which were very
definite and measured 5 to 7 centimeters across. Controls
with ‘“‘blastomycine’’ made in exactly the same way from
a blastomycecte isolated from a typical case of blastomycosis
did not give a positive reaction in this patient. Sporotricho-
sine injected into the skin of the patient with blastomycosis
gave no reaction; nor did he react to his own blastomycine.
Agar alone in 1% per cent suspension injected into persons
when taking potassium iodide (t. i. d. 10 grains) gave a
definite reaction but was not nearly as pronounced as that
given by the ‘‘sporotrichosine’’. Normal persons receiving
potassium iodide (t. i. d. 10 grains) reacted no stronger to

Beihefte zur Med. Klinik, Basel, 8, p. 179, 1909.

126 UNIVERSITY OF WISCONSIN STUDIES

sporotrichosine than before the iodide had been administered
to them. In either case the dermal reaction measured from
5 to 15 mm. whereas the sporotrichosine reaction in the patient
measured 5 to 7 em.

While, therefore, a striking reaction may be elicited in these
cases with sporotrichosine, it probably cannot be considered
sufficiently specific to be of value for differentiating these or-
ganisms. But it is to be noted that patients taking or not
taking potassium iodide may react intradermally to these
various strains of sporotricha and not to strains of blastomy-
ces prepared in exactly the same way. Whether or not all
cases will so react we do not know.

SuMMaRY AND DISCUSSION

It appears that the first case of sporotrichosis was reported
by Schenck in 1898. The second case was reported in 1900
by Hektoen and Perkins and the organism definitely identified
lv comparison with that isolated by Schenck. Hektoen named
the organism Sporothrix schencki.

In 1903 de Beurmann and Gougerot reported the first case
in France and the organism was named by Matruchot and
Ramond Sporotrichum beurmanni. The work of the Ameri-
can investigators published several years previously was not
known to the French workers.

On comparing the American strain sent to them by Hek-
toen in 1906, seven years after its isolation, de Beurmann and
Gougerot pointed out certain differences between this strain
and their recently isolated strain. They noted certain differ-
ences also between their own strains (strains a, b, ¢) but con-
tended that these differences were not sufficient to justify ere-
ating a new species. But the differences between their strains
and Sporotrichim schenchii were sufficient, they contended.
to justify a new species. The organisms, isolated later in
North America and those found in South America and in
Madagascar, they claim are the same as their organism, Sporo-
trichum beurmanni. These were all isolated after they dis-
covered and named the organism in France.

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 127

It is pointed out that sporotricha, French, American and
other strains, are especially subject to undergo pleomorphic
changes, some of which are transient while others are fixed
and permanent. De Beurmann and Gougerot themselves have
ealled especial attention to this and admit that some of the
pleomorphic alterations in the macroscopic growth of certain
strains render them identical with the strain of Sp. schenckii
as it exists today. Furthermore, Hektoen has stated that the
culture as sent to them had changed in the seven year interval
on artificial media especially in its ability to produce spores.
This change is not an uncommon one in both American and
French strains and no doubt was associated with other pleo-
morphic changes. Yet de Beurmann and Gougerot and also
Matruchot used this loss of ability on artificial media to form
spores as a differentiating characteristic from their own Sporo-
trichum beurmanni though they observed this same change in
strains of the latter. There can be no doubt that pleomor-
phic changes took place in Sporotrichum schenckii; and this
is borne out also by the photographs in both Schenck’s and
Hektoen’s papers which clearly show that at first both strains
produced spores in large numbers.

Changes in pigmentation are common in all strains of sporo-
tricha; poorly or non-pigmented strains may arise from deeply
pigmented strains and remain fixed. One would scarcely
classify such an organism as sporotrichum on the basis of such
a fluctuating character as pigmentation, though this property
is mentioned by them as an important differentiating one.

The statements of de Beurmann, Gougerot and Matruchot
that the Sporotrichum schencktt (original) does not form
chlamydospores must be considered erroneous. Under suitable
conditions these structures have been observed by the writer,
and these results were confirmed by Meyer, not only in the
original Schenck-Hektoen strain but in many other American
strains from both man and horses. The attempt to differen-
tiate Sporotrichum schencku and Sporotrichum beurmanni on
this basis must therefore be given up.

The fermentation of sugar is quite inconstant. It is diffi-
eult to understand why de Beurmann and Gougerot would use

128 UNIVERSITY OF WISCONSIN STUDIES

the differences in the fermentation of lactose and saecharose as
a distinguishing feature between the American and French or-
ganisms since they say that not all their strains fermented
saccharose. Meyer and Aird have shown conclusively the in-
constancy in fermentative powers of not only many American
strains but also of different French strains. They conclude
it is impossible to differentiate sporotricha into two distinct
species by means of the fermentation of carbohydrates. These
results have been confirmed by the writer. Greco’s observa-
tion on a South American strain does not agree with those of
de Beurmann and Gougcrot on French strains.

Specific serum and dermal tests are probably of limited
value in differentiating these closely related organisms though
they may furnish important data for the basis of a group
relationship. So far as the results indicate they show no dif-
ferences between the French and American strains.

From the above analysis it would seem that the basis upon
which French investigators differentiate the Sporotrichumn
schenckiit and Sporotrichwn beurmanni is, to say the least,
very inadequate. On account of the pleomorphism of this
organism there is an excellent opportunity to take advantage
of sight and unimportant differences in order to create new
species. It is of course true that no two strains are eractly
identical. De Beurmann and Gougerot noted that certain
of their strains manifested fixed pleomorphic changes that
made them appear identical with the original Sporotrichum
schenchii. Yet they did not suggest that these strains be
ealled Sporotrichum schenckti. Concerning these slight dif-
ferences, especially in organisms of this type, it would seem
that it would be wise to assume a conservative attitude and
to refrain as much as possible from the use of new and un-
necessary terms.

The question arises in connection with an organism of this
kind, as it arises so frequently in biology, what differences
are sufficient to warrant the creation of a new variety or
species? Where shall the line be drawn between varieties
since no two cultures of sporotricha are absolutely identical
in every dctail and strains are ever prone to these striking

IDENTITY OF AMERICAN AND FRENCH SPOROTRICHOSIS 129

changes? To this the answer must be made that this is
largely a conventional matter and often it is impossible to
state clearly where the line of demarcation should lie. But
this I wish to point out, that it is evidently not proper or scien-
tific to use pleomorphism, or any other character for that mat-
ter, as a basis for the classification of an early American
strain, and not use it in the classification of French strains or
later American strains.

In résumé, I believe we are justified in stating that the dif-
ferences between the American strains, including the original
cultures of Schenck and of Hektoen, and the French strains
of de Beurmann, Gougerot and others, are easily explained as
pleomorphic variations and therefore are insignificant. Fur-
thermore, the disease, clinically, pathologically, experiment-
ally, and therapeutically, is admitted by all to be identical in
France and in America.

The above statements being true, according to the rules of
botanical nomenclature the organisms in both countries should
be called by the name first given to them in 1900 by Hektoen,
namely, Sporotrichum schenckun. The fact that de Beurmann
rediscovered the organism several years later deserves no
consideration so far as determining nomenclature is concerned.
As regards the use of the compromise term Sporotrichum
schenckit-beurmanni, suggested first by Greco of South Amer-
ica and more recently concurred in by Meyer in this country,
it may be said that this is objectionable because it not only
introduces a long cumbersome term but it is not in accord
with the rules of botanical nomenclature. There is obviously
therefore but one legitimate term for this organism, namely
Sporotrichum schenckii.

It should be pointed out that even though one maintains
that the small differences noted between the pleomorphic
forms of the Schenck-Hektoen strain and the other sporotri-
cha are sufficient to justify a species distinction, the important
fact remains that the hundreds of strains of sporotricha
found in France and in North America are alike. This is
admitted by both sides of the controversy. Therefore, which-
ever view of the original Schenck-Hektoen strain is taken by

9

130 UNIVERSITY OF WISCONSIN STUDIES

the French, the identity of sporotrichosis, excluding the very
rare strains mentioned earlier in the paper, in France and
America must be admitted. One is as justified in making this
statement as in saying tuberculosis in France and in America
is identical.

THE SIGNIFICANCE OF YEASTS AND OIDIA IN
PASTEURIZED BUTTER ;

F. W. Bouska anp J. C. BRown

The original object of pasteurization in buttermaking was
to produce a better immediate flavor by controlling fermenta-
tions. The aim was to destroy undesirable microorganisms as
well as microorganisms whose efféct is unknown. By means
of a pure culture of selected lactic acid bacteria (starters),
regulated temperatures, and the acid test, the desired flavor
was to be developed. In more recent times it was discovered
that pasteurization greatly improved the keeping quality of
creamery butter made from cream which had soured spontan-
eously on the farm. A considerable part of American butter
is now made from such cream. The succession of sweet milk
by sour cream as a source of creamery butter is a result of
economic conditions on the farm. Some farmers think that
the expense of hauling milk to the creamery every day is too
great. So they skim the milk at home and deliver the cream
twice a week in the winter and three times a week in the sum-
mer. The skim milk thus derived has the best feeding value
because it is warm and sweet. But the cream obtained by this
farm method is usually sour. It became our lot to design
methods of making butter from spontaneously soured cream
and to overcome butter defects that sometimes occur here.
While our experience, investigations, and results apply prin-
cipally to sour cream, it will be seen that in some respects a
general application can be made.

Butter which has a good flavor when churned but develops
a bad flavor at low temperatures within one month has poor
keeping quality. Properly made pasteurized butter scoring
90 will, in current commerce, remain eatable to the last morsel.
In cold storage it scores 89 to 90 at the end of seven to ten
months. We have known churnings of butter to score 90 at
the end of eighteen months.

132 UNIVERSITY OF WISCONSIN STUDIES

A quicker test is made hy storing small samples at 60°
to 70° F. A very poor keeper develops a bad flavor within
three days. A good keeper, such as is usually made, scores
89 to 90 at the end ot two weeks.

It became our problem to ascertain the cause of poor keep-
ing and to find a remedy for it. Bacteriological philosophy
of the keeping quality of butter has exerted an exaggerated
influence in every sphere of butter-making. For a time
we accepted the impression from Sayre, Rahn and Far-
rand? that only yeasts can tolerate the well nigh saturated
solution of salt which is the watery part (brine) of butter.
To our disappointment, we did not find any connection be-
tween yeasts and a very long and serious epidemic of fishy
butter. Never present in great numbers, sometimes absent,
they practically did not multiply in the course of time.

At the beginning of 1913 we thought that by ascertaining
the number of yeasts and Oidia (lactis) in butter we could
foretell its keeping quality. We made a study of the cold
storage of 177 lots of butter from a number of states. As
the butter went into cold storage, the commercial judge made
his predictions according to the quality of the butter and
previous experience with the creamery. We predicted from
the number of veasts and Oidia. At the end of the storage
season the predictions were compared with the final quality
of the butter. We predicted that 39 lots would keep well.
Only five (13 per cent) of these kept poorly. The judge pre-
dicted that 83 lots would keep well. Twenty-four (29 per
ect) of these came out poor. But our way selected so few
good keepers that a vold storage could not have made a living
by our method.

We now know that under American creamery conditions
keeping quality is due to acidity, elimination of buttermilk,
pasteurization, and proper working, The deterioration of
butter is mainly the result of physical or biochemical causes.
An indirect part may be played by micro-organisms.

These bacteriological attempts taught us how to make counts
of yeasts and Oidia. We found that vatfuls and churnfuls

1 Technical Bulletin, No. 1, Michigan Experiment Station.

YEASTS AND OIDIA IN PASTEURIZED BUTTER 133

of raw spontaneously soured cream always contained from
10,000 to 100,000 yeasts (per ec. ¢.) and not quite so many
Oidia. In the winter the numbers are smaller with the Oidia
often preponderating. Properly pasteurized cream, right
from the pasteurizer, never contains yeasts or Oidia. Here,
then, is the test for pasteurized cream and pasteurized butter !
But cream off the cooler, in the cream vat, in the churn, and
the resulting butter, showed many veasts and Oidia. The
first and last cream issuing out of flash pasteurizers was not
pasteurized and had to be diverted by a by-pass till a pasteur-
izing temperature had been reached and maintained for sev-
eral minutes. The last cream had to be heated in a can or
held over till the next pasteurization. The outlet of re-
tarding and pasteurizing vats formed a long pocket in which
cream escaped pasteurization. This pipe was shortened. Raw
cream dripped into the pasteurized cream from pipes and
conductors that had conveyed the raw cream. These were
removed. Raw cream splashed upon the sides and cover of
a vat escapes pasteurization and is usually washed into the
churn with the hose. This is partly overcome by rinsing with
hot water or hot cream during pasteurization.

No amount of scrubbing of utensils or pipes will produce
good bacteriological results. This must be completed by
sterilizing by means of hot water or live steam. Permanent
steam connections on cream pipes give the best results. The
packing glands of vats, pumps, and churns and the wood of
churns, as they are today, are difficult to sterilize and furnish
some of the yeasts and Oidia. Sometimes the starter becomes
contaminated and introduces yeasts and Oidia. It is easy to
see how laymen handling both raw cream and pasteurized
products may contaminate the latter with their hands, ther-
mometer, dipper, and measuring stick.

In December, 1912, in one plant we began yeast and Oidia
counts in a eubie centimeter of butter. The totals ranged
from two to fifty. In August, 1913, the counts declined to
ten or less. We continued improving methods and equip-
ment and educating the men, and by July, 1916, we reduced
the counts to zero to five. This efficiency is now main-

134 UNIVERSITY OF WISCONSIN STUDIES

tained by 10 per cent of the plants; 20 per cent of the fifty
plants on which we have reeords are below 10. The majority
of them have less than six months’ education. As they learn,
we hope for better results. By means of written directions
and photographs, creamery workers are instructed how to
take samples of butter. (see Fig. 1.) They have proved quite
dependable. For transporting we use small metal capped

Fig. 1.—Soimpling butter for yeast and mold count.

vaseline jars sterilized in parchment wrappers. Butter is
nearly always sampled in the churn by means of a scalded
teaspoon. Prints and tubs are sampled by removing inner
portions by means of scalded knife, spatula, or trier. Enam-
eled or copper syrup pitchers are used for collecting a com-
posite sample of a day's churnines. The Hd on the pitcher
automatically drops down and prevents possible contamina-
tion.

Samples of salted butter sent by mail or stored for a week
do not show any multiplication. Unfortunately, yeasts and
Oidia multiply rapidly in unsalted butter. Counts of sweet
butter over 24 hours old are unreliable.

YEASTS AND OIDIA IN PASTEURIZED BUTTER 135

Whey agar is prepared as follows: Skimmilk is warmed
to about 100° F., acidified with lactic or hydrochloric acid,
coagulated with rennet or pepsin, the curd is cut, allowed
to settle and then heated to 115° C. in the autoclave, the
whey is filtered off through cotton, neutralized, made up
with 1.5 per cent agar, 1 per cent peptone, and filtered. In
plating, 1 ¢. ¢. of sterile 1 per cent tartaric acid solution (by
weight) is placed in the petri dish. Then 1 ¢. ¢ or 1 gram
of the material to be analyzed is introduced. With this is
mixed 10 ¢. ¢. of the agar. In two or three days at room
temperature the yeast colonies are about 3 mm. in diameter,

Fig. 2.—Yeasts and Oidia (Left) 1 ¢. ¢. of inefficiently pasteurized cream plated in
pened tartaric acid agar. (Right) In 1 ¢. ¢. of butter plated in whey tartaric acid
raised, moist, and glistening. The Oidium colonics are about
1 to 3 em. in diameter, dry and velvety. One soon learns to
recognize them at once with the unaided eve (see Fig 2).
Mixed colonies occur, but they do not affect the practical
interpretation of the count. Colonies of bacteria seldom
reach such a size as to interfere. Occasional air molds are
regarded as an accidental contamination unless they recur
in considerable numbers in the subject from the same source.
Professor Lund has suecessfully used beerwort and lactic
acid instead of whey and tartaric acid. Where obtainable,
the wort is more convenient than whey.

We have not made any study of the species or races of
yeasts and Oidia that occur here. We are unable to give any

136 UNIVERSITY OF WISCONSIN STUDIES

interpretation of a large proportion of yeasts and a small
proportion of Oidia and vice versa. Only the sum total has
a meaning. The yeasty are more persistent. To simplify
the language for laymen we call Oidium lactis ‘‘mold’’.

On the basis of the record made by a number of good plants
we have adopted an arbitrary commercial standard of ten or
less. This is as good work as the best men are able to do.
A count of thirty or more means one or more of the following
defects: Failure of pasteurization, i. e., in temperature, in
time, or by contamination; lack of cleanliness or of sterility of
utensils and conduits; or contaminated starter. In every case
where we made a personal survey where the count exceeded 30
we demonstrated that one of these defects existed to such
a degree that the laymen could easily see it when it was
pointed out to them. By mail and without making personal
trips we have corrected many defects. That we detected dirty
eream pipes 500 miles beyond our eyesight and caused them
to be cleaned is one of the wonders of our uninitiated.

When testing pasteurization or when searching for defects,
we present a survey of the plant by means of the graphic re-
‘port shown here (sce Fig. 3). This maps out the course
of the material through the plant and shows where defects
occur. In the case exhibited here yeasts and molds in the
pasteurized products made their first appearance in the churn,
thereby proving it to be the source of contamination. If the
cream were the original source of yeasts and molds, then the
butter would contain much fewer than the cream because the
drainage of the buttermilk aud washing of the butter eliminate
the larger part of them. But since the churn was the source
of the yeasts, their number increased the longer the materials
remained in the churn. The working of the butter expelled
more yeasts than the churning because the fixtures of the
worker are the main refuge of yeasts. These churns were
old and difficult to sterilize. Nevertheless this exhibit is ex-
cellent work and far above the average.

Other conditions being favorable, a butter having only a
few yeasts and molds is a safer hazard for long distance ship-
ments and for storage. Indeed, our records show that the

YEASTS AND OIDIA IN PASTEURIZED BUTTER 137

ereameries that have the best commercial reputation for their
butter also have the lowest yeast and mold counts. We must
acknowledge that in this case good all around methods con-
tribute to both results. At the plant where we made our
original study we have observations on a large part of its
stored make of 1912. Twenty-five per cent of this kept poorly.

No. 1 Receiving Yat

Aoidifiers 1,950,000 ,000
Putrefiers Count failed
0
Uolae. 307000 No. 4 First Cream out
Total 1,050 ,060,000 Yeasts None
| Molds None
No. 2 Pasteurizer Total
Acidifiers None
Putrefiers Count fetled
Yeasts” None
Molda None lo. 5 Last Cream out
Neutral 1,000
Total 1,000 —— | Yeasts None
| Molds None
No. 7 Vat Total
Yeasts Bone
Molda Bone
No, 6 Starter
Totel
——
Yeasts one
| Molds None
No. @ Churn (cream)
Total
Yeasts 2
Molds one
Total 2 Efficiency of pasteurizetion 99.99%
.No. 3 Butter |
Acidifiers 20,000
Putrefiers Count failed
Yeasts 2
Kolds 1
Total 20,003

Fig. 3.—Bacteriological Survey of the Creamery, May 15, 1917. By F. W.

Bouska.
The commercial storage referred to above is another case and
there 43 per cent of the butter did not keep. In 1915 at the
original plant 1.75 per cent of the storage spoiled. But in
1916 only 1/15 of one per cent did not keep. During this
time big improvements in every facility that affects keeping
quality were made. There is no way of ascertaining how much
credit for keeping quality should be awarded to each facility.
All men in any way associated with the making of this butter

138 UNIVERSITY OF WISCONSIN STUDIES

concur in giving a fair share of credit to the yeast and mold
counts.

The conditions of pasteurization that yield good results by
our test are considerably above the usual hygienic and board
of health requirements. Moreover, plating a whole e. ¢. of
butter gives a test one hundred times as exacting as plating
1/100 of ac. ec. Pasteurized butter yielding a low yeast and
mold count therefore has a clean bill of health. If butter that
purports to be pasteurized contains hundreds of yeasts or
molds, we would hesitate to say that the pasteurization that
produced it fulfilled hygenic or commercial requirements.
Nor could we positively say that it is lawfully entitled to be
branded ‘‘Pasteurized butter’’.

We cannot form even a conjecture of the meaning of large
or small numbers of yeasts and molds in raw butter. It
is not uncommon to find hundreds of yeasts and molds in
raw butter that enjoys the very best commercial reputation.

Our investigations and observations cover approximately
153,000,000 pounds of creamery butter.

THE ACTION OF CERTAIN BACTERIA ON THE
NITROGENOUS MATERIAL OF SEWAGE

KE. G. BirGe

Edward Grant Birge was born April 24, 1881, at Leip-
zig, Germany, at which time his father was studying at
the University of Leipzig.

Educated in the public schools at Madison, Wisconsin,
graduating from the high school in 1899.

Entered the University of Wisconsin in the fall of that
year where he took the premedical course, graduating in
1903.

Entered Johns Hopkins, graduating with the M. D.
degree in 1907.

For three years was bacteriologist with the Sewerage
Commission of the city of Baltimore, going from there
to Altoona, Pennsylvania, where he became bacteriolo-
gist for the Pennsylvania railroad.

In the fall of 1912 he went to Harvard Medical School
as assistant to Dr. Milton J. Rosenau in the department
of Hygiene and Preventive Medicine.

-In 1914 he went to Florida as State Bacteriologist
where he remained until the United States went into the
war.

In April, 1917, he volunteered with the Medical Re-
serve Corps and was given the commission of Captain.

He was sent to Fort Oglethorpe in August of that
year where he remained for nearly a year. From there
he was sent to Camp Beauregard, Louisiana, and then to
Camp Wadsworth, Spartanburg, North Carolina. He re-
ceived his discharge in August, 1919, going immediately
to the University of Iowa as Professor of Bacteriology
and State Epidemiologist, which position he filled until
his death from influenza on February 4, 1920.

The bio-chemical work which has been done on sewage in
the past has been confined almost entirely to the changes
taking place in the various forms of filter beds. We have
then considerable information concerning bacterial action in
the filter beds, but our knowledge of this action in the septic
tank is in a more chaotic state. What little we know is the
‘“mass action”’ of all organisms, bacterial and otherwise, which
play a role in the preparation of sewage for further treat-
ment.

The primary object of this paper, which must be considered
as preliminary, is to determine what certain individual species

140 UNIVERSITY OF WISCONSIN STUDIES

of bacteria do chemically when allowed to act on sewage in
pure cultures singly or mixed. It was hoped that something
could be shown which would justify us in trying to control
the bacterial flora in large masses of sewage under treatment.
It was also expected that the results would enable us to say
what we might expect if certain groups or species of bacteria
proved to be present in predominant numbers.

It is reasonable to assume that mere numbers of micro-or-
ganisms do not guarantee any definite chemical action. It is
only when certain species or groups are predominant that a
definite chemical change may be predicted or expected. At
the present time we can make no prediction of that sort, be-
cause it is not known what bacteria or groups of bacteria are
responsible for those changes which are considered desirable.

In the past, work on putrefaction and decomposition has
been confined largely to the study of the nitrogenous material,
although there are certain other cycles which, as Fuller’ points
out, must be quite as carefully studied. In conformity with
past work along this line, it was decided to study the nitrogen
cycle.

Clark,” in his report on a somewhat similar investigation
done at the Lawrenee Experiment Station in Massachusetts,
points out that there are five lines of action, i. e., putrefaction,
nitrification, de-nitrification, nitrogen liberation, and nitro-
gen fixation. While undoubtedly all five of the processes go
on simultaneously, the process which predominates depends
entirely upon conditions present. His work was done in con-
nection with the nitrification in filter beds, but it is reasonable
to suppose that much the same thing would hold true for
septic tanks, except that the conditions are such, usually, that
putrefactive action is more likely to predominate, especially
in the one story type of tank.

At the beginning of this work it was assumed that the most
likely change in composition would be an increase in the free
ammonia content, and a corresponding decrease in those de-
composition products represented by the organic nitrogen,

1Fuller, Sewage Disposal, p. 47.
?Clark, Report of the Mass. State Board of Health, p. 244, 1904.

THE ACTION OF CERTAIN BACTERIA ON SEWAGE 141

with little or no change in either the nitrites or nitrates.
In order to bring about conservation of the higher oxidation
products of nitrogen, it is essential that sufficient oxygen be
present so that the micro-organism is not forced to reduce
these substances to support life. I believe that at the present
time it is considered wise to have as much residual oxygen
as possible in the septic tank. I found, as I will point out
later, that the action of certain bacteria, in fact of nearly all
of the particular species studied, was much more dependable
under aerobic than anaerobic conditions.

METHODS

Tlie methods used simulated natural conditions as much as
possible in laboratory experiments on a small scale. In order
to use sewage as a culture medium it is necessary to sterilize
it, and any method of sterilization changes the chemical com-
position of the sample. It is therefore necessary to select
that method which produces a minimum change. Chemical
methods are naturally out of the question, because of their
ineffectiveness in bringing about complete sterility without a
great excess of the chemical, which would have an inhibiting
action on the bacteria subsequently added. The incomplete-
ness of sterilization by chemical means has been shown by
Lederer and Hommon? in their paper before the American
Public Health Association, at the meeting of 1910.

If the gross solid material is filtered out, the sewage can
be sterilized by heat, without greatly affecting the chemical
composition.

While there is a small difference in composition between
the unsterilized and sterilized sewage, the differences in the
flasks of sterilized sewage made up from the same large
sample are so slight as to be negligible.

The sewage used was filtered through cotton to remove
the gross solid material, put into liter flasks and sterilized at
120° C. (15 pounds pressure) in the autoclave for 45 minutes.
Two sets of flasks were made up at a time for each series. To

> Lederer and Hommon, Journal American Public Health Association, I
(New Series), p. 267, 1911.

142 UNIVERSITY OF WISCONSIN STUDIES

one set, paraffin oil was added to give anaerobic conditions
and one was left without the oil to favor aerobic conditions.
In each set there was one flask which acted as a control. The
changes in those flasks which were inoculated with bacteria
were compared with this control, after making allowances for
difference in composition and errors in methods.

In eonnection with the dissolved oxygen content, the pre-
liminary work brought out several interesting facts. It was
found that the heat of sterilization would in itself drive off
the greater amount of the dissolved oxygen, but that the flasks
which were not treated with paraffin oil would pick up oxygen
rapidly, even at the temperature of the incubator, 37.5° C.
On the other hand those which had been treated with paraffin
oil continually lost what little dissolved oxygen there was left.
There is no doubt but that the unsaturated hydrocarbons of
the oil extracted the dissolved oxygen, and did it so rapidly
that at the end of twenty-four hours there was not, for all
practical purposes, any left.

The flasks were inoculated twenty-four hours after steriliza-
tion with 1 ¢. e. of a twenty-four hour bouillon culture of the
bacteria to be studied and incubated at 37.5° C. for twenty-
four hours. They were then analysed for free ammonia, or-
ganic nitrogen, nitrites and nitrates according to the Standard
Methods for Sewage Analysis.

Sewage.—The sewage was obtained from the Ward Street
Pumping Station of the Metropolitan System of Boston, and
was a typical domestic sewage. The district served by this
pumping station was mainly residential. There was probably
a small amount of manufacturing wastes and some storm
water. Care was taken to obtain the samples at the same time
each day, so that the sewage would have a similar composition
throughout the work.

Bacteria.—The bacteria chosen for the work were not iso-
lated from the sewage itself. It was thought better to use
bacteria of known stock, typical of those groups which are
normally found in sewage. For this reason B. coli, B. cloacae,
B. pyocyaneus, B. proteus, B. mesentericus var. ruber and
B. subtilis were selected. All of these have been reported as

THE ACTION OF CERTAIN BACTERIA ON SEWAGE 143

having been isolated from or are representative of groups
normally occurring in sewage.

The bio-chemistry of these bacteria, when grown on artifi-
cial media, shows that they possess characteristics which,
theoretically at least, should be of value in breaking down a
complex organic substance such as sewage. The only excep-
tion to this statement is possibly the power of B. coli to reduce
nitrates. It will also be recalled that all of them are faculta-
tive anaerobes.

The action of the bacteria when grown in mixed cultures
brought out two. very interesting points. One was the behavior
of B. coli. Invariably in the anaerobic flasks it was com-
pletely overgrown in the twenty-four hour period. Under
aerobic conditions it overgrew B. pyocyaneus, but was over-
grown by other bacteria.

The other fact was that both B. proteus and B. subtilis over-
grew the other bacteria in the proportion of three to one
under all conditions. When these two were grown together
they about balanced each other at the end of the twenty-four
hour period.

THE ACTION OF PURE CULTURES

The action of pure cultures was studied under two conditions
of temperature, at 37.5° C. and at room temperature. A
summary of the results follows:

B. coliThere was a marked tendency to decrease the
nitrites and nitrates, with a very decided decrease in the
organic nitrogen. This was more marked in the anaerobic
flasks than the aerobic flasks, and was much more vigorous
at 37.5° C. There was very little action on the free ammonia,
although the general tendency was to decrease it.

It was noted that the correction for the amount of organic
nitrogen added to the sample in the bouillon tended to give,
in all of the experiments, a reduction of the organic nitrogen.
This was studied in the hope that some intermediate decom-
position products might be found to account for it. At the
present time I am unable to explain it in any other way than

144 UNIVERSITY OF WISCONSIN STUDIES

that the standard methods may tend to give results too low,
and that possibly some of the nitrogen is liberated.

B. cloacae.—In general it may be said that this micro-or-
ganism did not materially differ from B. col. The greatest
action was on the nitrites and nitrates, which were decreased
markedly in both the anaerobic and aerobic flasks. There was
also a decided tendency to decrease the amount of free am-
monia, although this was not constant.

B. pyocyaneus.—At 37.5° C. the action was irregular. At
times the nitrites and nitrates were increased and under ap-
parently the same conditions they were decreased. The
same holds true for the free ammonia and organic nitrogen.
There was nothing to indicate that the oxygen content of the
samples had any effect on the action.

At room temperature the action on the free ammonia and
organie nitrogen had the same irregular tendencies that were
noted above.

This irregularity was also noted in the aerobic flasks in
connection with the nitrites and nitrates. In the anaerobic
flasks the nitrates were decreased with an increase in the ni-
trites. This increase and decrease nearly balanced.

B. proteus.—This also showed a marked irregularity in its
action at 37.5° C. At room temperature the action was more
constant, the free ammonia being increased with a correspond-
ing decrease in the organic nitrogen. The nitrates were in-
creased with a corresponding decrease in the nitrites. This
activity was more marked in the anaerobic than in the aerobic
flasks, and I believe that with this micro-organism, better
results can be obtained under anaerdbic, or nearly anaerobic
conditions, than where the oxygen content is relatively high.

B. mesentericus var. ruber.—This has nearly the same
characteristics as the previous micro-organism, the action being
irregular in the experiments conducted at 37.5° C. while
those at room temperature were more constant, especially
under anaerobic conditions. At the lower temperature the
free ammonia was increased and the organic nitrogen was
decreased. The nitrites were, in the majority of the experi-
ments, increased, with no changes noted in the nitrates. Under

THE ACTION OF CERTAIN BACTERIA ON SEWAGE 145

aerobic conditions at room temperature, both the nitrites and
nitrates were increased, while the organie nitrogen was de-
creased. These changes did not balance, leaving a large defi-
cit in the organic nitrogen which has not been satisfactorily
accounted for.

B. subtilis—This micro-organism gave really consistent
results. They were obtained under aerobic conditions and
confined themselves to the free ammonia and the organic
nitrogen. There was in all of the experiments an increase in
the free ammonia with a decrease in the organic nitrogen that
practically balanced it. This activity was relatively very
great, amounting to as much as eight parts per million
at 37.5° C. and two parts per million at room temperature.
In regard to the nitrites. and nitrates, the tendency was more
often to decrease than to increase them, but this action was
not constant.

Under anaerobic conditions the action of the bacteria was
regular at 37.5° C., both the free ammonia and the organic
nitrogen being decidedly decreased with an increase in the
nitrites and nitrates, the latter being very decided. At room
temperature the action was not so marked and in general
followed that of the aerobic conditions, showing an increase
in the free ammonia and a decrease in the organic nitrogen.

From the results which were obtained, I believe that this
micro-organism and the entire group of allied micro-organisms
will bear much careful study in their relation to the bacterial
processes in the purification of sewage.

The action of mixed cultures.—I have already pointed

out that in mixed cultures B. coli was completely overgrown
in the anaerobic flasks, and way overgrown by all of the bac-
teria in the aerobic flasks, with the exception of B. pyocyaneus.
Also that B. subtilis and B. proteus (vulgarus) overgrew the
other bacteria in both sets of flasks, When grown together they
about balanced each other.

The fact that the chemical action very closely followed
the predominating bacteria was extremely intcresting. The
action was in certain cases, as I will point out later, very dif-
ferent in the aerobic and anaerobic flasks with the same

10

146 UNIVERSITY OF WISCONSIN STUDIES

bacteria. This difference was particularly noticeable when
B. subtilis and B. coli were grown with other bacteria.

It was to be expected that the chemical results of these ex-
periments would follow in a general way the influence of the
predominating bacteria, although the action of the other or-
ganisms was at times apparent. The time of incubation was
in some cases evidently too short for the predominating bacter-
ium to manifest itself chemically. This was plainly evident
where more than two bacteria were inoculated into one flask.
In these cases also it is to be noted that B. cola dropped out
of sight in twenty-four hours in both sets of flasks. In those
flasks where all of the bacteria were grown together the over-
growth of B. proteus and B. subtilis was apparent, but it was
not sufficient to markedly affect the chemical results. A sum-
mary of the results follows:

B. coli and B. cloacae.—I have pointed out previously
that these two organisms have almost. the same action on
sewage. The results when grown together are quite different
from what might be expected, especially in the aerobic flasks.
Here the free ammonia was increased with a corresponding
decrease in the organic nitrogen. The nitrites were also de-
creased. No changes were noted in the nitrate content. Ap-
parently the presence of B. coli was a decided advantage in
the early part of the incubation period, since it was greatly
overgrown in twenty-four hours.

The overgrowth in the anaerobic flasks was probably much
quicker than in the aerobic flasks, and was much more com-
plete. The chemical changes noted in this set of flasks are
those of a pure culture of B. cloacae, the only exception being
that neither the nitrites or nitrates were attacked.

B. coli and B. mesentericus var. ruber.—The changes
here are those due almost entirely to B. mesentcricus var.
ruber, especially in the anaerobic flasks.

In the aerobic flasks the small number of B. coli present af-
fected the results so that they were very irregular.

B. coli and B. pyocyaneus.—In spite of the fact that
B. coli overgrew B. pyocyaneus, there were no changes which
could be attributed to either micro-organism. Evidently B.

NS

THE ACTION OF CERTAIN BACTERIA ON SEWAGE 147

colt established itself so late in the incubation period that
the little chemical effect it might have had was not apparent.

B. coli and B. proteus.—In pure culture both of these
bacteria had a slight and irregular chemical action. However,
when grown together, the changes in composition were regu-
lar and consistent. In both the aerobic and anaerobic flasks
there was an increase in the free ammonia and a decrease in
the organic nitrogen. No changes were noted in either the
nitrites or nitrates.

B. coli and B. subtilis—In the aerobic flasks the changes
were those of a pure culture of B. subtilis, 1. e.,
an increase in the free ammonia and a corresponding de-
crease in the organic nitrogen. Except in one case where there
was a slight increase in the nitrites, neither the nitrites or
nitrates showed any change.

As I have noted, neither of the bacteria has a regular
chemical action at 37.5° C. under aerobic conditions. How-
ever, when grown together there is an increase in the free
ammonia and a decided decrease in the organic nitrogen.
This increase and decrease balanced each other.

Neither the nitrites nor nitrates were affected in any way.

Just what the explanation for this condition is, I am unable
to say at the present time, as B. cola was completely overgrown.

B. subtilis and B. cloacae.—In the aerobic flasks, in which
B. subtilis markedly overgrew B. cloacae, the chemical
changes were typical of B. subtilis. The free ammonia was in-
creased with corresponding decrease in the organic nitrogen.
The nitrates were decreased with an increase in the nitrites.

In the anaerobic flasks where the overgrowth of B. subtilis
was not apparent, the chemical changes were in the main those
of B. cloacae, differing from those of the pure culture in the
tendency towards increasing the nitrites and nitrates.

B. subtilis and B. mesentericus var. ruber.-—In the aerobic
flasks the action was irregular, resembling that of B. mesen-
tericus var. ruber. Both the nitrites and nitrates were in-
creased.

In the anaerobic flasks the free ammonia was increased
with a corresponding decrease in the organic nitrogen and

148 UNIVERSITY OF WISCONSIN STUDIES

nitrites. The nitrates were unaffected. This, except for the
decrease in the nitrites, follows very closely what was found
under similar conditions for B. mesentericus var. ruber in
pure cultures. The cultures, however, showed a considerable
overgrowth of B. subtilis.

B. subtilis and B. pyocyaneus.—In both sets of flasks the
bacteria grew practically equally well.

In the aerobic flasks the free ammonia aud the organic
nitrogen were decreased, while the nitrites and nitrates were
both increased.

In the anaerobic flasks the free ammonia was increased
with a deercase in the organic nitrogen and nitrites. The ni-
trates were unaffected.

B. cloacae and B, mesentericus var. ruber.—B. mesenteri-
cus var. ruber overerew B. cloacae in both sets of flasks.

In the acrobic flasks the chemical action was more irreg-
ular than was found for B. mesentericus var. ruber in pure
culture, but in general coincided very closely to it. The most
marked effect was on the nitrites and nitrates. The nitrates
were decreased with an inerease in the nitrites.

Tn the anaerobie flasks the action was much more typical
of B. mesentericus var. ruber, the free ammonia being in-
creased with a decrease in the organic nitrogen. The nitrates
were decreased and the nitrites were inereased.

B. cloacae and B. proteus.—The action of these bacteria
was very irregular and typical of neither. This was true for
both the anaerobic and aerobic flasks.

The nitrites were generally inereased. Jn one experiment
all of the nitrogen was reduced in amount.

B. proteus and B. mesentericus var. ruber.—In the
anacrobie flasks the tendeney was to decrease both the free
ammonia and the organie nitrogen, while the nitrites and
nitrates were increased, These results follow those obtained
from B. proteus more closely than B. mesentericus var, ruber.

The cultures showed a slight overgrowth of B. proteus in
the anaerobic flasks.

B. pyocyaneus, B. proteus, B. pyocyaneus and B. mesen-
tericus var. ruber.—Neither of these gave results in either
set of flasks.

THE ACTION OF CERTAIN BACTERIA ON SEWAGE 149

The chemical results in those experiments in which four
of the bacteria were inoculated into one flask were very ir-
regular and disappointing. The time of incubation was ap-
parently insufficient for any one or any combination of the
bacteria to establish themselves chemically.

In the experiments in which all of the bacteria were in-
oculated into one flask there was but one change worthy of
note. That was the increase in the nitrates in all of the ex-
periments. This was found under both aerobic and anacrobie
conditions,—amounting in one instance to six parts per mil-
lion. There was a decided decrease in the rest of the nitrogen
present.

SumMaRY AND CoNCLUSIONS 4

Certain bacteria have been studied to determine their effect
upon sewage. Those selected were B. coli, B. cloacae, B. pyo-
cyaneus, B. vulgaris, B. mesentericus var. ruber, and B. sub-
tilis. They were selected because they represent aerobic types
which are found frequently or constantly in sewage. The
effects of these bacteria were studied in fresh sewage, filtered
and sterilized by heat at 120° C. (15 pounds pressure) for
forty-five minutes. They were studied under aerobic and
anaerobic conditions, also in pure and in mixed cultures.
Particular attention was paid to the changes in free ammonia,
organic nitrogen, nitrites and nitrates.

Bacteriologically it was shown that B. coli was completely
overgrown under anaerobic conditions in the twenty-four hour
period. Under aerobic conditions it was able to overgrow B.
pyocyaneus, but was overgrown by the rest of the bacteria
studied.

When grown in pure cultures, with the exception of B.
proteus, the bacteria gave more constant results under aerobic
than anaerobic conditions.

B. subtilis showed a marked ammonifying power through-
out the work under aerobic conditions. Under anaerobic con-
ditions it regularly decreased the free ammonia and organic
nitrogen content, increasing the nitrites and nitrates, especi-
ally the latter.

150 UNIVERSITY OF WISCONSIN STUDIES

B. coli and B. cloacae had a decided reducing action on
both the nitrites and nitrates. Under some conditions, as yet
undetermined, they reduced the free ammonia content also,

B. proteus had a considerable ammonifying power under
anaerobic conditions. This was very slight under aerobic
conditions, and was more constant at room temperature than
at 37.5° C.

The action of B. pyocyaneus and B. mesentericus var. ruber
was irregular under both aerobic and anaerobic conditions.

The experiments with mixed cultures showed that the
chemical changes followed very closely those of the predomi-
nant bacteria in pure culture. Those experiments which did
not follow this rule, and in which there was a decided pre-
dominance of one micro-organism, showed that the bacteria
had become predominant too late in the incubation period to
effect a chemical change.

B. coli and B. cloacae, B. coli and B. proteus gave more
constant results when grown in mixed than when grown in
pure cultures. These were the only instances of an appar-
ently true symbiotic relationship.

The results of the experiments in which more than two
bacteria were grown in mixed culture showed that the in-
cubation period, twenty-four hours, was too short to allow any
one micro-organism of any group of bacteria to establish it-
self chemically.

The work has shown that we will be able to predict what
the changes in the chemical composition of sewage are going
to be if a group or species of bacteria are predominant.

The results of this work would certainly not justify us
in attempting to control the bacterial flora of the septic tanks.
However, I am strongly convinced that in the future this
course will be attempted.

I again wish to emphasize the results obtained from B.
subtilis, Everything indicates that this organism may be
made to play a most important réle in the treatment of sew-
age.

THE DETECTION OF PASTEURIZED MILK
W. D. Frost

The necessity for pasteurizing all milk destined to be used
as human food is becoming more and more firmly established
ag the dangers from the use of raw milk are more generally
recognized. But in order to further safeguard the public
health it is necessary to control the methods of pasteurization.

The range of temperatures permissible in pasteurization is
very narrow. In order to render a milk safe it must be heated
above the thermal death point of Bacillus tuberculosis. Only
a few degrees above this necessary temperature the physical
properties of milk are altered. The cream line is changed and
a ‘‘cooked taste’? may be acquired. Both of these changes
lessen the commercial value of the milk. Hence there is con-
stant temptation for the milk dealer to underheat his milk.
Public health authorities must therefore be constantly on
the alert to prevent the sale of underheated or improperly
pasteurized milk.

How determine whether or not milk has been properly
pasteurized? So far as I know the milk analyst is helpless
unless he makes use of the method discussed here.

It is true, of course, that various methods have been sug-
gested. One of these proposes to regulate the temperature
and time of holding in pasteurization. To this end certain
municipalities require the use of automatic thermoregulators
and recorders on all pasteurizing apparatus. A bacteriological
test tells whether or not a milk is high or low in bacteria, but
cannot always, by any means, indicate the thoroughness
of the process of pasteurization. Other tests have been advo-
cated which depend upon the changes which the protein under-
goes in heated milk. These have not proved applicable in
practice. Still others have been suggested which depend
on the presence of oxidizing enzymes in milk. Of these,

152 UNIVERSITY OF WISCONSIN STUDIES

Storch’s test is generally regarded as the most satisfactory.
This test, however, as is well known, can be used only on milk
heated from 78° to 80° GC. (172.4° to 176° F.). It cannot,
therefore, be applied to milk heated to the temperature em-
ployed for pasteurization in this country now. A microscopic
test was devised by me and described in a paper by Frost
and Ravenel in 1911.4. Two features of this method inter-
fered with its usefulness. One was the difficult technic and
the other was the fact that the stain safranin clotted the milk
unless added with the greatest care. So far it has been pos-
sible to avoid this danger only by diluting the stain, and in
this case the action of the dye is largely neutralized.

In 1915 I proposed the method under discussion in this
article in a preliminary paper. This was described somewhat
more in detail in a paper read before the International Milk
Dealers’ Association in October, 1916.2 The practical results
that may be obtained by its use are described in a paper by
Miss Moore and me.?

It is here proposed to discuss this method in a somewhat
more adequate way.

Bopy CELLS In MILK

There occur in cow's milk under all conditions a variable
number of body cells or leucocytes. There are at least two
kinds: the mononuclear and the polymorphonuclear varieties.
The latter ones are of chief interest here. Whether or not
these cells are leucocytes from the blood which have escaped
through the gland walls or are cells from the walls of the
mammary gland, given off at the time of milking, is a ques-
tion on which histologists are not agreed.

1 Frost and Ravenel, A Microscopie Test for Heated Milk in Proc, Am.
Assn. Med, Milk Comanissions, p. 127, 1911.

2W. 1). Frost, A Microscopic Test for Pasteurized Milk in Jour, Am.
Med. Assu., 44, pp. 821-822, March 6, 1915.

3°W. D. Frost, Milk Dealer, p. 84, Dec., 1916.

*W. D. Frost and Genevieve D, Moore, The Cellular Test for Pustewr-
dared Milk in Dairy Science.

THE DETECTION OF PASTEURIZED MILK 153

Lacking definite evidence to the contrary and in keeping
with common practice, these cells will be referred to as leu-
cocytes. The variety which is of service in this study is
histologically described as follows:

Polymorphonuclear or polynuclear leucocytes constitute the
form more frequently encountered (in normal blood), and prob-
ably represent the fully developed condition of the white blood-
cell. These elements are distinguished by the variously distorted
nucleus, which, being fairly rich in chromatin, presents a striking
figure in stained preparations. The nuclei appear imperfectly
separated into variously disposed segments, so that they recall the
letters O, S, U, V, Z, etc. The segments usually retain connec-
tion by delicate threads of chromatin; exceptionally these bridges
become broken, in which case the term polynuclear is appro-
priate. Occasionally cells may be observed containing granules
which stain deeply with eosin. Such eosinophilic leucocytes
probably represent the final phase of development.’

PRINCIPLES INVOLVED

Wet process.—In attempting to differentiate between
raw and pasteurized milk, I have® shown that when certain
stains are put into milk and allowed to act wet, there is a
distinct difference in the way in which the stain acts on the
cells in the pasteurized milk and on the cells in the raw milk.

When the proper amount of stain is used, the cells in the
raw milk are not stained at all, while the nuclei of the cells in
the pasteurized milk are well stained. If the stain should be

‘too concentrated, the nuclei of the cells in the raw milk may
be stained, but only slightly, while those in the pasteur-
ized milk are always deeply stained.

The staining of the pasteurized cells and the failure to
stain the cells in the raw milk is to be explained on the theory
that the heat of pasteurization is sufficient to ‘‘fix’’ (in the
histological sense) the protoplasm of the leucocytes and thus
make staining possible.

Brief description—The method may be briefly described
as follows: The milk to be tested is mixed with an equal

5 Pierson, Textbook of Normal Histology, sixth edition, p. 106.
® Loe. cit. ,

154 UNIVERSITY OF WISCONSIN STUDIES

quantity of a special methylene blue stain in a specially pre-
pared centrifuge tube. The stain and milk are thoroughly
mixed and allowed to stand for at least ten minutes. The
sediment is thrown onto the cork by whirling the tube
in a centrifuge. The sediment is carefully spread on a micro-
scopical glass slide. This smear is allowed to air dry with-
out coming in contact with water at any time. When dry
it is examined under the microscope with a low power and
later with the oil immersion objective.

DetaiLs or MretHop

Preparation of stain—Various stains have been tested
out: safranin, methylene green, dahlia, thionine and methylene
blue. On the whole methylene blue seems to be the stain
best adapted to the test. Practically all of the work has been
done with the German dye (Gruebler), but several American
made dyes have been tested recently and have proved to be
entirely satisfactory.

At first quite concentrated solutions were used. Small
quantities of these were added to larger volumes of milk.
_ While this method produced satisfactory results, there was a
tendency for these dyes to curdle the milk, and when this
happened the test was worthless.

I have found gradually that this danger can be overcome
by using a more dilute stain and adding larger proportions
of it to the milk. It is quite essential to the proper working
of the test that the right amount of the dye be added to the
milk. Fortunately this is indicated by the color of the
milk. When methylene blue in any concentration is gradually
added to milk the color becomes a deeper and deeper blue, un-
til quite suddenly it becomes a ‘‘cadet blue’’. This deep blue
color seems necessary to the proper working of the test. It
may be arrived at in any way, but there is less danger of
causing a precipitation if the dye is added to the milk in a
weak solution.

To make the solution needed, 1.5 grams of the methylene
blue powder is added to a liter of distilled water. I have

THE DETECTION OF PASTEURIZED MILK 155

usually allowed this to stand in the 37° C. incubator for sev-
eral hours (over night) and then filtered it through paper.
Such a stain when added to milk, part for part, should pro-
duce a ‘‘cadet blue’’. Some powdered dyes may need to be
used in greater concentration, others, especially the medicinal
methylene blue, in smaller quantities.

This solution seems to be quite permanent and most of my
evidence indicates that it keeps indefinitely.

Mixing stain and milk.—The addition of the dye to the
milk can be done in any convenient way. At first I put
the milk in a flask or beaker and added concentrated stain
slowly while the milk was kept moving by a rotary motion
of the vessel. When a weaker solution as recommended above
is used, the method of combining them is quite immaterial
except that it is never safe to drop the milk into the dye. It
is better to add the dye to the milk. The mixing should be
thoroughly done.

Time of action of stain.—The action of the stain requires
several minutes and it has been my practice to mix the stain
with the milk and then allow it to act for ten or fifteen minutes
before it is centrifuged. A longer action up to one hour is not
injurious, but if the stain is allowed to stand too long in con-
tact with the milk the cells of the raw milk may take on the
stain, but even under these conditions a careful study of such
cells would enable one to recognize the difference between
them and the cells of properly pasteurized milk.

Centrifugation—Any form of centrifuge tube can be
used for collecting the sediment. The most convenient are
those which have a rubber stopper at the lower end because
of the ease with which the sediment can be removed. At first
I used the Stewart-Slack tubes, but lately I have made a
larger tube of the same style. In this way I always get
an abundance of cells for examination, and avoid the use
of a special head on the centrifuge. The tubes are made from
three-eighth inch glass tubing and are four inches long. One
end is plugged with a rubber stopper (No. 00), the other is
left open. These tubes can be put into the ordinary head of
any centrifuge. The centrifuge should be run long enough

156 UNIVERSITY OF WISCONSIN STUDIES

and at'sufficiently high speed to throw down most of the leu-
cocytes without injuring them, e. g., 2,000 r. p. m. for ten min-
utes.

Spreading of sediment.— When the tubes are taken out
of the centrifuge, the fat layer at the top of the tube is
loosened, the milk and stain mixture poured out, the cork re-
moved, and the sediment on it carefully spread on a glass
slide, as one would make a blood smear. It must always be
berne in mind, however, that water cannot be used to spread
the smear, as this violates the principles involved and in-
variably causes the cell nuclei to take up the stain, even in
raw milk.

Microscopical examination of smears.—The smears arc
first examined with the low power (16 mm. lens). In the
raw milks the background, or the entire microscopic field,
is stained blue, the depth of the stain depending upon the
thickness of the film: in this blue background appear numerous
clear areas. The smaller of these are fat globules. The larger
ones may be large fat globules or clusters of the same, but
usually they are the leucocytes of the milk, Occasionally
there are the deeply stained mononuclear leucocytes. The
general impression one gets from these preparations is a blue
field, which tends to be uniform, and in which there are a
number of holes or clear places. :

The smears of the pasteurized milk, on the other hand, do
not have the background as deeply stained as do the smears
from the raw milk, although in thick portions it may be quite
blue. The nuclei of the leucocytes here are always stained and
their volor is deeper than that of the background. The area
immediately surrounding the cells usually takes the stain
deeply and shades off into the color of the background, forming
what I have called a ‘‘dark halo’’.

Because of the lighter color of the field, the fat globules
do not stand out as they do in the raw milk. The most
prominent objects here are the leucocytes with their darkly
stained nuelei.

The cells are noticeably smaller in the pasteurized than
they are in the raw milk.

THE DETECTION OF PASTEURIZED MILK 157

Even a cursory examination of the two smears shows.a dis-
tinct and easily recognizable difference between the smear
from the heated milk and that from the raw milk. __

To examine the cells more closely, the oil immersion ob-
jective is used. The oil may be put direetly onto the dried
smear, or the preparation may be mounted in Canada balsam
under a cover glass.

The polymorphonuclear leucocytes in the raw milk,
under the high powers of the microscope, are practically all
colorless, but with some experience it is not difficult to
recognize them. Usually they are quite regular in outline
and large, i. e., about twelve microns in diameter. The nu-
clear material, if differentiated at all, is poorly defined and if
stained it is of a light greenish-blue color. Occasionally
there are leucocytes in raw milks, and in some more than
others, which take the stain. Whether or not these are
dead cells has not been determined. Even if quite deeply
stained the leucocytes in raw milk are distinetly different
from those in pasteurized milk, in that the nuclear
material is more spread out and less densely stained. The
nuclei of the raw cells are usually integral with a definite
isthmus connecting the lobes.

The leucocytes from properly pasteurized milks have their
nuclei deeply stained, and the different portions are frequently
rounded up into definite fragments so that the cells appear
to be polynuclear rather than polymorphonuclear. They aver-
age about seven microns in size. The depth of the stain and
the amount of shrinking vary somewhat with the degree of
heat applied.

By way of summary it may be repeated that:

The effect of heat on the leucocytes, so far as this test is
concerned, is twofold. It alters the shape and size of the cells,
and changes their staining reactions. The shape of the cell is
probably gradually changed as the degree of heat increases, and
the shrinking begins to appear at a lower temperature than that
used for pasteurization; but the ‘‘fixing’’ of the nuclear material.
which makes possible the absorption of the stain, seems to take
place definitely at practically the same temperature as that nec-

158 UNIVERSITY OF WISCONSIN STUDIES

essary for the pasteurization of milk, namely, at from 60 to 63°C,
(140-145°F.) 7

INTERPRETATION OF RESULTS

Illustrations: The results of this method of staining are
shown in Plates I and II, Figures 1 to 26. These drawings
are all made under an oil immersion lens and by means of
the camera lucida.

Figures 1 to 4 show cells in the raw milk. Here the back-
ground is stained, while the cells remain clear or unstained.
In Fig. 5 the milk was heated to 58° C. (137° F.) for 20
minutes and in Fig. 6 to 60° Co. (140° F.). Here the poly-
morphonuclear leucocytes have their nuclei lightly stained.

Figures 7, 8, and 9 show the cells in milk that has been
pasteurized at 63° C. (145° F.) for 20 minutes. The nuclei
are all deeply stained and the segments well rounded off and
the fragments discrete. Fig. 10 shows cells from milk heated
to 65° C. (150° F.), and Fig. 11 shows cells from milk
heated to 70° C. (158° F.). Figures 10 and 12 show the
bacteria well stained.

Figures 13 to 18 represent groups of leucocytes selected
from various fields to show variations in form, size, and
staining reaction at the various temperatures indicated. Ex-
eept for the raw milk, no attempt has been made to represent
the background.

Figures 19 and 20 are the results obtained by applying a
counter stain to the preparations obtained by the staining
with methylene blue in the usual way. The advantage of the
counter stain is that it brings out or differentiates the leu-
cocytes in the raw milk. (The preparations are made in the
usual way and when the smears are dry, they are immersed
for a few seconds in a 1 per cent solution of orange G in
aleohol (95 per cent) and examined after drying without
washing. )

Figures 21 and 22 are from preparations made by a method
suggested by Traum.® Jn this method the cells are separated

‘ Loc. cit.

®J. Traum, Laboratory Methods to Improve Milk in Amer. Jour, Vet.
Med, 10, 717, 1915.

THE DETECTION OF PASTEURIZED MILK 159

by centrifugal force, smeared on slides, dried, and fixed with
formalin. The staining of this dried, fixed film is done
with a one-half saturated aqueous solution of methylene blue.
The nuclei of the cells in the raw milk stain well (Fig. 21).

They appear as if drawn out and there is frequently no indi-
cation of cell body, the area around the nuclei fusing into the
background. If a cell body is observed it has a reticulated ap-
pearance. In pasteurized milk, on the other hand, the nuclei of
these cells are smaller, more compact, stain deeper, and the cells
which are considerably smaller, are outlined by a clear, circular
zone around the nucleus. The difference is noticeable when com-
paring raw milk with milk heated to 60° for 30 minutes and is
very marked if the milk has been heated to 63° for 30 minutes.

Figures 23 to 26 represent preparations made from raw
milk (23), pasteurized at 58° C. (24), 60° C. (25), and 63° C.
(26) and stained with Wright’s blood stain. The milk sedi-
ment was spread on slides, dried, and then stained in the
usual way.

Derails oF PROCESS

Stain of background.—The body of the smear, probably
the casein or milk serum, takes the stain in a varying degree.
In raw milk the background is usually found to be dark and
continuous. By this is meant that the entire smear is stained
deeper than the nuclei of the leucocytes which are imbedded
in it. Usually these are not stained at all. Whether it is
actually dark or light depends very evidently upon the thick-
ness of the film and this unfortunately cannot be controlled.
In well pasteurized samples the stain of the background is
found to be light and variable. By this is meant that the
stain in the background is lighter than the nuclei of the con-
tained leucocytes. It is variable in that it is usually darker
about the leucocytes, forming a more or less distinct ‘‘dark
halo’’ about them.

Stain in cells—In raw milk the leucocytes are unstained
and in the typical preparations appear as clear areas in a
dark blue field. They are easily differentiated from the fat glob-
ules by their larger size and more or less irregular outline.

160 UNIVERSITY OF WISCONSIN STUDIES

Frequently there is a tinge of blue to them so that there is
just a suggestion of the nuclear structure of the cell visible.
A few cells even in perfectly fresh milk may have their nuclei
stained but less densely and compactly than in pasteurized
milk. In the pasteurized milk, on the other hand, the nuclei
are definitely stained, usually dark blue, while the background
is lighter. The difference is striking and perfectly charac-
teristic.

Outline of leucocytes.—The outline of the cells in raw
milk is irregular. This is probably due to protrusions or
pseudopodia although certain authors have hesitated to accept
these eells as leucocytes because they do not show evidence of
aineboid movement. The irregular outlines, as shown in the
figures of raw milk, are noticeable, however, especially in
Figures 4 and 13.

In the heated milk the outline of the cells usually appears
more regular. Under the influence of heat the cells have
rounded up. There is no constant difference between these
cells under the two conditions, however, and some observers
have even regarded the heated cells as the more irregular.
The irregularity of the heated cells may, however, be due to
quite a different cause, namely, shrinkage.

Nuclear fragments.—Only the polymorphonueleated cells
are of value in this test. In a raw cell it is usual for the nu-
cleus to be single but variously shaped (or polymorpho), while
in the heated milks there is a pronounced tendeney for the
nuclei to become separated and thus the eclls become poly-
nucleated. The nucleus or its fragments are much more
compact and demarkated in the heated than in the raw samples.

Size of the leucocytes.—<As already pointed out, the heat
of pasteurization not only affects the staining reaction of
the leucocytes but it produces a profound change in their size.
In raw milk these cells are frequently 10 or 12 microns in
diameter and nearly always above 7.5 microns. In properly
pasteurized inilks they are very much smaller, usually less
than 7.5 microns.

This marked reduction in size is no doubt duc to the heat
of pasteurization which shrinks the cells. The decrease in
size is a progressive one, depending upon the amount of heat

THE DETECTION OF PASTEURIZED MILK 161

used. When a temperature of 63° C. (145° F.) is maintained,
the average diameter of the cells is only slightly greater than
half the diameter of the cells in raw milk. It is evident from
Figure 14 that the shrinking is less at 60° C. (140° F.) than
it is at 63° C. The shrinking continues with the increase
of heat. See Figures 15 to 18.

The table below gives the comparative size of the milk cells
in raw and pasteurized samples. All of the figures given are
the average of at least twenty-five cells.

SIZE OF MILK CELLS

Raw Pasteurized
11.2 5.6
9.8 6.0
10.4 7.5
10.9 7.0
12.9 6.4
11.9 7.2
11.8 6.8
11.0 6.2
13.0 5.7
12.0 6.0
Grand average 11.4 6.4

Temperature at which change takes place.—The staining
reaction typical of properly pasteurized milk occurs only
when milk hag been subjected to a particular temperature
for a definite length of time. With the time constant, say
thirty minutes, no change takes place in the leucocytes until
the temperature nears 60° C. It is true that the nuclei begin
to absorb the dye at temperatures from 56 to 60° C. At first
the nuclei stain only lightly and there is an almost invariable
tendency for them to be greenish in color even at 60° C. It
is usually possible to differentiate milk pasteurized at 60° C.
from that pasteurized at 63° C. and above, by the way the
nuclei take up the stain. It is only when a temperature of
63° C. is reached that the nuclei become deeply stained. Be-
yond this temperature there is no change in the character of
the stain although the nuclear fragments may be more com-
pact and discrete and the size of the cell reduced. The definite

11

162 UNIVERSITY OF WISCONSIN STUDIES

change in staining reaction between the leucocytes of raw
and pasteurized milk takes place at the temperature of 63° C.

When milk is subjected to this temperature for varying
lengths of time it is found that the definite staining reaction
takes place when the temperature is maintained for about
twenty minutes. When the time of exposure is eighteen min-
utes or less, some of the leucocytes do not take the stain in
the characteristic way. So it may be stated that the character-
istic staining reaction occurs when the milk is subjected to
a temperature of 60° to 68° C. for upwards of twenty minutes.

Effect of stain on bacteria.—The heat which fixes the
nuclei of the leucocytes for staining curiously renders the
bacteria less likely to take the stain. It is only in raw milks
then that the bacteria stain well. In well pasteurized milks
they stain poorly. Frequently the bacteria take the stain with
a varying degree of intensity, but whether this is due to in-
herent differences in the bacteria or to the effect of the heat
on them is undetermined. The reaction of the bacteria to
the stain has up to now shown illogical variations, but in
general the following conclusions seem warranted. The
bacteria in raw milk are always stained, usually well and
sometimes very beautifully. In the sediment from properly
pasteurized milk examined within a few hours after pasteuri-
zation the bacteria are usually invisible. Old pasteurized
milk may show the nuclei of the cells well stained and the
bacteria also. In this case we assume that the bacteria which
stain have been introduced or grown subsequent to the pro-
cess of pasteurization.

THe MicroscoricaL PICTURE OF A PROPERLY PASTEURIZED MILK

When a milk has been stained by the Wet Process described
in this paper and the sediment mounted without the addition
of water is examined under the microscope, the following
picture will be obtained if it has been pasteurized at a tem-
perature of 63° C. (145° F.) or above, and held at that
temperature for twenty minutes or more. The nuclei of prac-
tically all of the leucocytes are in compact masses. They
are well stained. The background is lighter than the nuclei

THE DETECTION OF PASTEURIZED MILK 163

except in the thickest places. The leucocytes are usually sur-
rounded by a ‘‘dark halo’’, They are small,—not more than
7.5 microns in diameter. If the sample is examined soon after
pasteurization or has been kept under conditions which pre-
vent the growth of bacteria, the bacteria will not stain at all
or at least variably or indistinctly.

In making this examination the slide is ordinarily first ex-
amined under the low power, and the light, uneven distribu-
tion of the background and the deeper stained leucocytes are
noted if the milk has been properly pasteurized. In a raw
milk the background is quite blue in the thicker places and
the unstained leucocytes appear as clear areas in it.

The slide would ordinarily next be examined under an
oil immersion objective. A few leucocytes would be studied
to determine their staining reaction and the condition of the
nuclear fragments. The eyepiece should be provided with
a micrometer scale and some of the leucocytes measured. The
condition of the bacteria will be noted without effort at the
same time.

THE Use oF OTHER STAINS

A number of the anilin dyes have been tried out in this test,
as for example safranin, thionine, methylene green, dahlia,
gentian violet and methylene blue, but none of them appear
to possess advantages over methylene blue.

THE INVESTIGATION OF DRINKING WATER
SUPPLIES

H. A. WHITTAKER

The question of what constitutes a safe water supply for
drinking purposes, and the proper method of determining
this fact are subjects that have given sanitarians much con-
cern ever since the discovery of water-borne diseases. In-
vestigators have worked for many years developing and per-
fecting field and laboratory methods for the detection of un-
safe supphes, devising corrective measures to eliminate the
dangers found to exist, and preventing the repetition of these
errors in new installations. This work has resulted in the
discovery of certain general principles concerning the pro-
tection of water supphes, and in establishing analytical
methods and standards for their control. The application
of these principles, methods, and standards to water supply
investigations by different health organizations has differed
widely throughout the country. The water supply service
has often been adapted to the existing health organization
rather than the organization being adjusted and equipped
properly to handle the work. Some organizations have fol-
lowed the dangerous practice of depending largely upon an
analysis of the water without requiring a detailed field survey
by a trained observer to secure accurate information regarding
the location, construction, and the management of the supply.
A common practice is to leave the ficld survey of the supply
to untrained individuals in the local communities. Data
sheets and sampling equipment are often furnished to local
authorities and private citizens who collect the field data and
water samples on which the safety of the supply is determined.
There are several points in connection with this practice that
are dangerous to public safety. The field survey, which is
one of the most essential parts of the investigation, is placed

INVESTIGATION OF DRINKING WATER SUPPLIES 165

in the hands of an untrained observer who is often incompetent
to undertake the work. This same unskilled individual is
entrusted with the duty of securing samples of water that
must be properly collected if satisfactory results are to be
obtained. These samples are then shipped to the laboratory
and subjected to a most careful examination by a skilled
technician when there is little assurance of the accuracy of
their collection. This method makes it necessary for the skilled
worker to accept facts from an untrained person on the funda-
mental features of an investigation on which the safety of
a water supply is to be judged. The fallacy and danger of
such practice is very evident when these points are considered.
The importance of thorough investigation work on water
supplies is illustrated in the following tables. These tables
include six years of investigation work on existing water sup-
plies by the Minnesota State Board of Health.

TABLE I.-WATER SUPPLY INVESTIGATIONS, 1912-1918

| Results
Water Supplies Investigated

Satisfactory | Unsatisfactory

Number ssus6ss0)sjeiainserianeiaesncicrelawens laleeges 1,119 389 730
Per cent ......... sears uy aa apes 100 34 66

The investigations recorded in Table I represent both sur-
face and underground water supplies from a variety of
sources including wells (dug, bored, drilled, driven), springs,
lakes, rivers, creeks, etc. This table shows that 1,119 existing
water supplies were investigated during the period indicated.
Three hundred eighty-nine, or 34 per cent, were shown to
he safe and 730, or 66 per cent, were found to be unsafe
sources in their existing condition.

TABLE IL—UNSATISFACVrORY WATER SUPPLIES, 1912-1918

Shown Unsatisfactory by

Unsatisfactory Water

Supplies Field survey and Field survey Analytical results
analytical results
Number ...... .......6+ 730 354 | 338 | 38

Per cent ........0.0-e05 160 49 46

166 UNIVERSITY OF WISCONSIN STUDIES

Table II includes the unsatisfactory supplies found during
the same period. An analysis of this table covering the un-
satisfactory supplies shows that 354, or 49 per cent were found
to be unsafe by both the field survey and the analytical re-
sults; 338, or 46 per cent, by the field survey alone, while
the analytical results on the date of the investigation were
satisfactory ; and 38, or 5 per cent, by the analytical results
alone where the field survey did not show the possibilities for
pollution and further investigation was required to find the
avenues through which pollution could enter the supply.
These results show that the field survey was corroborated by
the analytical results in 49 per cent of the cases; that the
field survey was the only index of danger in 46 per cent; and
the analytical results the only indication in 5 per cent. These
results demonstrate the importance of thorough field survey
work, for had the analytical results been accepted as the
only index, 49 per cent of the unsatisfactory supplies would
have been approved. It is also true that if the analytical
work had been omitted, 5 per cent of the unsatisfactory sup-
plies would have been overlooked.

It is evident from these results that both field and analytical
investigations should be made in order to determine the safety
of a water supply from a sanitary point of view. It should
be appreciated that the use of any method which permits the
collection of haphazard information by untrained individuals
is exceedingly dangerous.

The field survey should give an accurate idea of the possi-
bilities of present and future pollution. The analytical re-
sults should provide information on the sanitary condition
of the water at the particular time the investigation is made
and it may supply some information of its past history. The
ficld survey and analytical results combined should afford
information on which recommendations can be made for the
protection ov the abandonment of an unsafe supply. The cor-
rective recommendations are frequently very simple and a
water supply can be made safe at a relatively small cost.

In order to illustrate the importance of thorough investiga-
tion work, especially in regard to the ficld survey, it may be

INVESTIGATION OF DRINKING WATER SUPPLIES 167

of interest to enumerate some of the most common errors that
are found associated with water supplies. The errors referred
to are briefly summarized as follows:

1) The use of surface waters without treatment, as all
such waters must be considered open to contamination. The
sanitary policing of such sources is- usually impossible or im-
practical.

2) The installation of water purification plants in localities
where underground supplies were available and would have
provided a safer source.

3) The installation of water purification plants by local
authorities who have little or no understanding of the theoreti-
eal and practical side of the treatment nor the difficulties
attending the operation of these plants. Many such plants
have been installed and the authorities believed them to be
equipped with automatic devices which would insure an ef-
fluent of good sanitary quality. This impression is erroneous
since these plants require the most careful attention by trained
operators at all times during the operation.

4) The installation of hypochlorite or liquid chlorine plants
to purify water that cannot be properly purified by these
chemicals bécause filtration is required to render it suitable for
such treatment.

5) The addition of hypochlorite or liquid chlorine to raw
water as it enters a filtration plant rather than to the effluent.
This practice often makes very difficult the administration of
these chemicals in proper amounts.

6) The failure to keep on hand duplicate parts of im-
portant equipment, especially parts of chlorine apparatus that
are likely to get out of repair. The lack of such parts may
make it necessary to allow untreated water to be discharged
into the distribution system.

7) The installation of by-passes around water purification
plants by which untreated water can be admitted into the
system, without passing through the plants.

8) The improper operation of water purification plants
by unskilled or inefficient operators.

168 UNIVERSITY OF WISCONSIN STUDIES

9) The location of wells, pumping apparatus, exposed
suction mains, reservoirs and filters where they will be subject
to flooding with surface water.

10) The improper construction of well casings and covers,
and the lack of adequate provision for surface drainage to
prevent pollution of the well with surface water.

11) The construction of pits around wells at the surface
in which all or a part of the pumping equipment is located.
This applies especially to drilled wells. It is difficult to pre-
vent surface and waste water from collecting in such pits.

12) The connection of any part of the system with sewers
which makes it possible for sewage to back up into wells, well
pits, storage reservoirs, etc.

13) The improper construction of underground and sur-
face reservoirs. This includes structures that are not water-
tight, those where the covers admit surface water and others
where the clean-outs are connected with sewers or bodies of
polluted water, the surface of which may be raised to an eleva-
tion higher than the bottom of the reservoir.

14) The installation of emergency connections which may
permit untreated surface water, or water of unknown or ques-
tionable sanitary quality, to be admitted to the system. There
should be a complete physical separation between water sup-
plies that are safe and those that are known to be unsafe from
a sanitary point of view.

Many of thege defects have been the cause of epidemics of
water-borne diseases, consequently, their sanitary significance
is at once apparent. These errors can be prevented in new
supplies and corrected in existing supplies by proper health
administration carried out by trained workers who have the
proper facilities to put such supervision into effect.

CONCLUSION

The purpose of this article is to draw the attention of
health authorities and others to the importance of thorough
investigation work when the safety of a water supply is to be
determined from a sanitary point of view. The investigation
should be undertaken by trained workers who understand the

INVESTIGATION OF DRINKING WATER SUPPLIES 169

proper application of methods, the interpretation of results,
and by those who are competent to make definite reeommenda-
tions for correcting defects or for the abandonment of a water
supply. The use of haphazard methods leads to erroneous
opinions or false security which may result in loss of life
among the consumers.

THE MILK SUPPLY OF CHICAGO
A. L. Amorr

Source oF SUPPLY

The city of Chicago is very fortunately situated in that it
is located in the heart of one of the most fertile agricultural
sections in the United States. No other large city is so favor-
ably situated, with the exception, perhaps, of Milwaukee,
where almost identical conditions prevail.

As the city grows and expands,-we find the dairy district
gradually moving farther from the city limits, to make room
for new subdivisions, suburban towns, and to a great extent,
truck gardens.

During the years 1907 to 1911, 90% of the milk reaching
Chicago was produced within 56 miles of the city. Since that
time the milk zone proper has moved out about 25 miles.
This has reference to the regular supply in normal times.
In case of a shortage in the Chicago milk zone, due to un-
usual or abnormal conditions, it quite often becomes necessary
to go considerably farther, as is the case at the present time,
when a small percentage of milk and cream is brought in a
distance of 275 miles.

The accompanying map is designed to show the growth
and expansion of the milk territory within recent years. Line
No. 1 shows the Chicago milk district up to 1915, as authorized
and inspected by the Chicago Health Department. Line No.
2 represents the district from 1915 to 1917, which was also
inspected by the Health Department. The district ineluded
between Lines 2 and 8 is not inspected by the Health Depart-
ment, although considerable milk is shipped into the city
from this territory.

In spite of the abnormal conditions prevailing at the present
time, brought on by the war, Chicago is the only large city

THE MILK SUPPLY OF CHICAGO

getting the bulk of its milk supply within 100 miles.

171

This

is all the more remarkable when it is borne in mind that no
supply can come from the east on account of Lake Michigan.

“EAU CLAIRE
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AMILWAUKEE GRAND RAPIDS
ie LANSING
“7

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fe” ft

8 «

ee ert.

LOGANSPORY WAY

sevarayi tte
s ey

PEORIA

ILLINOIS

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INDIANAPOL!9

Map Showing the Expansion of Milk Territory within Recent Years.

In the district, which from its geographical position, natur-
ally should supply Chicago, there are 42 condenseries, with a
combined capacity of 2,447,000 Ibs. of milk per day, which
divert the flow from its regular channels toward the city.
This is one of the contributing causes, if not the only reason,
why it has become necessary for the distributors of market
milk to seek other sources of supply in Wisconsin, Indiana,

Michigan, and to a less degree in Iowa.

172 UNIVERSITY OF WISCONSIN STUDIES

AMOUNT OF SUPPLY

There is a total of approximately 17,000 farms producing
milk for the city market. According to the most recent milk
‘census taken by the Chicago Health Department in August
and September, 1917, there are approximately 800,000 quarts
reaching Chicago daily, which includes that used for retail,
wholesale and industrial purposes. According to the popula-
tion of Chicago, this means an average per capita consump-
tion of .6 pint.

The Monthly Crop Report issued at Washington, D. C.
eredits the entire state of Illinois with 1,057,000 cows in 1917,

an increase from 1,047,000 in 1916. It is very evident that
Illinois produces many times the amount of milk consumed by
Chicago.

Sixty per cent of the total milk of Illinois’, ninety per cent
of Wisconsin’s and seventy-six per cent of Indiana’s are made
into butter, cheese and condensed milk.

McHenry County, Illinois, is the third largest market milk
producing county in the United States, being exceeded by St.
Lawrence and Orange Counties, N. Y. Kane County, Ilinois,
is the second milk producing county in Illinois and fourth in
the United States. Kane County borders on Cook County,
in which Chicago is located, and McHenry County borders on
Kane County, which gives an idea of the proximity of the
milk eenter to the city of Chicago.

It has been determined by the Chicago Health Department
that McHenry County, Illinois, and Walworth County, Wis-
consin, produce sufficient milk to supply Chicago with its
needs. According to the same authority the average number
ot cows per farm in 1916 was 1714 and in 1917, 1414; also
the average production per cow in 1917 was 16 lbs. per day.

The consumption of 800,000 qts. per day by the city of
Chicago is a subnormal figure, brought about to a certain ex-
tent by the propaganda of economy, and the increased price.
Decreased consumption in the city means a surplus in the
country which must be conserved. The Chicago zone is
practically devoid of butter and cheese factories, making it

THE MILK SUPPLY OF CHICAGO 173

necessary for the condenseries to absorb this surplus, which
sometimes works a hardship on that industry.

PRODUCTION

At the present time 98 per cent of the milk consumed in
Chicago is pasteurized, according to the Chicago Health De-
partment. The other 2 per cent is certified milk. A clause
in the city ordinance provides for the sale of raw inspected
milk, but none is sold, unless certified milk is included under
this head.

All pasteurized milk has been heated in a ‘‘holding system’’
since 1914. A minimum temperature of 140° F. with a min-
imum time of 20 minutes is required. Previous to 1914, the
“‘Hash’’ system was used almost exclusively.

The largest dealers in the past have advertised the fact
that their milk was bottled in the country, and at the present
time, this practice is maintained by them, although one of
the largest companies has erected and operated a modern plant
within the city limits for the past four years.

The plants located in the country are known as “‘bottling
plants’’. They are situated as a rule near a railroad track.
The milk is delivered at these plants, by the farmers them-
selves, between 6:30 and 11 A. M., depending on the season
of the year. In summer deliveries are made early, and
in winter late. It is a ruling of the milk distributors that
the milk cans be covered with canvas during transit from farm
to plant. ;

The milk is cooled by the farmer to 60° F. or below, usually
by placing the cans in a tank of cold running water. After
delivery at the plant, the cans are thoroughly washed and
steamed before they are returned to the farmers. Immed-
iately after delivery, the milk is pasteurized and cooled to
45° F. The milk is then filled into glass bottles; no single
service packages are used to any extent. All milk for the
retail trade has been bottled since 1891, and that for the
wholesale trade is put up in 8 or 10 gallon cans.

Some of the bottling plants are equipped with separators
and separate their own cream for the trade. The skim milk

174 UNIVERSITY OF WISCONSIN STUDIES

obtained is -utilized for the manufacture of buttermilk,—any
surplus is sold back to the farmers or made into casein.

The milk is put up in quarts and pints; cream in 14 pints,
and buttermilk in quarts. After bottling, the bottles are
placed in erates, covered with cracked ice, and transferred
to refrigerator cars for transportation to the city.

The milk ears reach the city not later than midnight of
the same day that they are loaded, under normal conditions.
They are switched to side-tracks controlled by the milk com-
panies, where they can be unloaded directly into delivery
wagons for distribution. There are 47 such stations owned
by the larger companies.

The big dealers order their milk from the bottling plants
daily, estimating very closely the amounts needed from day
to day.

The small dealers receive their milk from farmers designated
as ‘‘shippers’’. Their milk is shipped in 8 or 10 gallon cans,
in ordinary baggage cars, the shipper paying the freight.
These cars are switched to sidings and platforms owned by
the railroad companies. From there the cans are hauled by
trucks and teams to the various pasteurizing establishments of
which there are 305 in the city.

TRANSPORTATION

The city milk supply comes in over 25 different railroads
and electric lines. By far the greater portion is hauled by
the Chicago & Northwestern R. R. and Chicago, Milwaukee
& St. Paul R. R.

The freight tariff now in force is practically the same one
in effect during the last 20 years. The rates as between dif-
ferent railroads are practically the same for equal distances.

The milk put up at the bottling plants, or in cans at re-
ceiving stations, is brought to the city by what is known as
‘‘milk trains’. These trains carry nothing but milk, and
operate under normal conditions within a radius of 80 or
85 miles of the city. At the present time trains of this char-
acter are operating as far as 150 miles from the city, although

THE MILK SUPPLY OF CHICAGO 175

kin small quantities is brought from points 275 miles away.
e milk sent in by the ‘‘shippers’’ to the small dealers is
idled in baggage cars, attached to the passenger trains under
yedited movement.

[he railroad companies are not compelled by law to ice the
*s, consequently this is done by the milk dealers. The
hipper’’ is also obliged to furnish his own ice.

The freight rate varies, of course, with the distance, size
ean and material in the can, which the following table will
istrate :

Milk Milk or Cream |Cream and Condensed Milk
8 gal. 10 gal. Crate of 12
cans cans qts., 24 pts., 8 gal.eans | 10 gal. cans

48\% pts. or less

ke Geneva, Wis.....] 18.5¢ 02 25E 10.5¢ 23¢ 26¢
tamore, Ill. ........ 17.5¢ 21.5¢ 10.5¢ 23¢ 26¢e
vodstock, Ill. ...... 17.0¢ 21.0¢ 10¢ d 23¢ 26¢
st Chicago, Ill..... 15.5¢ 19.5¢ 9¢e 19¢ 21e

All empty bottles, cans, crates and boxes are returned to
iginal point of shipment free of charge. This is also true
th sour milk provided it is in the unskimmed condition.

City DistRIBUTION

The milk in Chicago is delivered almost exclusively by means
one horse wagons of which there are about 3,286 in number.
mmparatively few, if any, motor driven vehicles deliver milk
the consumer. One reason perhaps is the short haul between
yuses, which would make the starting and stopping of a
otor vehicle a hindrance rather than a help in efficient dis-
ibution.

Aceording to information furnished by the Chicago Health
epartment, there are in the city at the present time, 70
tablishments which deliver milk, but buy it already bottled,
)5 hottled milk establishments, 2 wholesale only establish-

176 UNIVERSITY OF WISCONSIN STUDIES

ments, 5 who deal in bulk milk only, and 21 wholesale bulk
establishments.

Of late there has been considerable discussion as to the
efficiency of the present system of distribution. Owing to
the fact that several dealers may have become firmly estab-
lished in practically the same section of the city there neces-
sarily must result considerable duplication and criss-crossing
of milk routes of these several distributors. Remedies have
been suggested, such as the division of the city into zones,
allotting certain sections to each dealer, or by the establish-
ment of municipal distributing stations under the direct super-
vision of the city authorities. Neither proposed system has
been practically applied. The establishment of milk depots
has been suggested, which would permit the customer calling
for his own milk, and eliminate the expense of delivery. The
latter scheme in all probability would not meet with success,
except perhaps in the poorer districts, due principally to
the fact that the public has been educated to the present sys-
tem and is especially loath to make early morning calls for
breakfast milk, if it is possible to have it delivered at the door.

This was one phase of the subject under discussion during
a recent milk inquiry conducted by the Food Administration
during December, 1917, and January and February, 1918.
From testimony brought out by experts on accounting and
cost figures, it would appear that any attempt to change the
present system of delivery would involve a considerable lapse
of time and expenditure of money.

PRICES

During the past two years, there has been considerable agita-
tion and discussion relative to the price of milk to the con-
sumer. During April, 1916, the first raise in price for several
years, from 8¢ to 9¢, caused considerable comment among the
public and press. When, however, the price was raised by
subsequent steps to 13¢ per quart, the consumer in consequence
cut down on consumption to a considerable degree.

An effort has been made to determine the relation between
increase in price and consumption of milk. The accompany-

THE MILK SUPPLY OF CHICAGO 177

ing chart shows a composite graph of several companies’ ex-
perience during the period of raise in price. The price of milk
was raised from 10¢ to 13¢ the first of October, 1917, and
lowered to 12¢ per quart on November 1st, 1917, and remained
stationary throughout November and December.

100,00

wal \ PAL
NCO

=

85,000 a 7
00
o 7 lw 1 25 | & 22 29 +6 3

OCTOBER NovemBER =‘ DECEMBER

The figures on the left of the chart illustrate the number of
points sold. A basis of 100,000 points was chosen as an
arbitrary figure and the graph shows the decrease on that basis.
The figures at the bottom represent the end of weeks. It may
be necessary to explain the meaning of the word ‘‘point’’.
A point is considered a quart of milk, or two pints of milk,
one-half pint of cream or a quart of buttermilk. We may,
therefore, assume that the figures on the left represent quarts
of milk.

There was a sharp decrease in consumption when the milk
was raised from 10¢ to 13¢ per quart. After November
Ist consumption gradually began to rise, although it did not
approach normal.

From an investigation conducted by several milk companies,
it was found that the greatcr percentage in reduction of
consumption of milk due to increase in price took place in the
poorer sections of the city. Similar investigations disclosed
that a very small percentage resorted to the use of canned milk.

12

178 UNIVERSITY OF WISCONSIN STUDIES

The general decrease in consumption has been estimated at
about 20 per cent, although the price of milk has not kept
pace with the price of a majority of other important articles
of food, during the present time of stress.

Previous to the year 1912, milk was bought from the farmer
at a flat rate per hundred weight. In 1912 a bonus was paid
for milk averaging 3.8 per cent fat for the month, which
marked the beginning of payment for milk on the butter-fat
‘basis. In 1915 this more equitable method of payment was
finally established. A price was fixed for milk testing 3.5 per
cent fat with an increase of three cents per hundred pounds
for every tenth of one per cent above 3.5 per cent and a sim-
ilar decrease of three cents per hundred pounds for every
tenth of one per cent below 3.5 per cent down to 3 per cent.

The following prices have prevailed in the Chicago milk dis-
trict for the last ten years. From 1915 on the prices are
based on 3.5 per cent milk with increase and decrease as noted
above. Previous to 1915 a flat rate per hundred pounds was
paid, regardless of butterfat tests.

oe) ford = a ical} ec) has] "D c= ~ he

&| = a| & &| sa Sel oes = me? oe

Month Ce ahe | fie oe) el) oe

5) 2/2 /2/2)] 2/2/2|] 2 &
ADT istiedigsissewsis 1.30 | 1.35 | 1.40 | 1.45 | 1.30 | 1.30 | 1.60 | 1.60 | 1.45 | 1.65 ) 2.40
MAY Seactesearniase 1.00 05 | 1.10 | 1.20 | 1.10 | 1.05 | 1.40 | 1.40 | 1.20 | 1.45 | 2.00
June -90 | .95 | .95 | 1.05 | 1.00 | 1.00 | 1.30 | 1.25 | 1.10 | 1.25 | 1.60
DUIY™ caavees sede qoek 1.00 | 1.05 | 1.05 | 1.20 | 1.30 | 1.30 | 1.50 ) 1.50 | 1.30 | 1.55 | 2.12
August .......... 1.20 | 1.25 | 1.25 | 1.30 | 1.45 | 1.45 | 1.60 | 1.60 | 1.55 | 1.70 | 2.380
September ...... 1.30 | 1.35 | 1.35 | 1.40 | 1.45 | 1.50 | 1.60 | 1.60 | 1.55 | 1.70 | 2.30
October .........) 1.55 | 1.45 | 1.55 | 1.70 | 1.70 | 1.60 | 1.85 | 1.90 | 1.53 | 1.90 | 3.42
November ...... 1.65 | 1.55 | 1.70 | 1.90 | 1.85 | 1.75 | 1.95 | 2.00 | 1.65 | 2.10 | 3,22
December ........] 1.65 | 1.55 | 1.75 | 2.00 | 1.90 | 1.80 | 2.00 | 2.00 | 1.65 | 2.10 | 3.22
January ........ 1.65 | 1.55 | 1.75 | 2.00 | 1.85 | 1.75 | 2.00 | 1.95 | 1.64 | 2.05 | 3.22
February ..,...-. 1.65 | 1.55 | 1.70 | 1.90 | 1.80 | 1.70 | 1.85 | 1.85 | 1.54 | 2.00 | 3.07
March ...-. eee 1.55 | 1.45 | 1.55 | 1.70 | 1.65 | 1.60 | 1.75 | 1.75 | 1.43 | 1.85 | 2.90

The prices beginning with November, 1917, were set by the
Food Administration during their hearing on milk prices
which will be referred to later. It has been customary to

THE MILK SUPPLY OF CHICAGO 179

sign a contract for six months with each individual farmer
for his supply during these months.

ORGANIZATION

The farmers in the Chicago district are members of the
Milk Producers Association. This Association came into
prominence about two years ago, although previous to that
time local organizations existed of very little importance to
the industry as a whole. Since the organization of this Asso-
ciation, numerous strikes or boycotts have been instituted
on the part of the farmers over the price question, resulting
in partial milk famines in the city. During the winter of
1917-1918, this matter became so serious that the United States
Food Administration started an inquiry to determine the cost
of production and distribution, in an effort to establish a just
and equitable price to the farmer and likewise to the consumer.

The producers refused to abide by the findings of the Com-
mission and instituted a boycott or ‘‘strike’’. The State Food
Administrator called another conference establishing a price
satisfactory to the producers.

The drivers of the milk delivery wagons are well organized
in the Milk Wagon Drivers Union. Their organization regu-
lates the hours of labor and wages of its members. The wages
are fixed at $21.00 and $22.00 per week, in addition to a com-
“mission of six-tenths cents per point over 1,333—14; points sold
per week or about 191 points per day. They are also entitled
to two weeks’ vacation every year with pay.

There exists also an organization known as the Milk Dealers
Association, its purpose being to promote the welfare of the
milk industry, especially as regards distribution. Local condi-
tions have made it possible for a firm of milk brokers to
develop. This firm serves to establish an equilibrium between
supply and demand. If a farmer or group of farmers wish
to dispose of their milk at some outlying point, the broker en-
deavors to find a market and also if a dealer in the city has
an over-supply the broker will also make an attempt to remedy
this condition.

180 UNIVERSITY OF WISCONSIN STUDIES

CertirieD MILK

Certified Milk was first introduced in Newark, N. J., orig-
inating with Dr. Coit. Gradually other communities adopted
this system of producing safe raw milk, and it was finally in-
troduced into Chicago in 1909. At this time the Chicago Medi-
eal Society appointed a Medical Milk Commission to certify
the milk from certain farms. At the present time eight dif-
ferent farms are sending Certified Milk to Chicago, four
located in the northern part of Illinois and four in the south-
ern part of Wisconsin.

MILK INSPECTION

From 1890 to 1900 great stress was laid on the fat side of
milk. From 1900 to the present time, the pressure which
has been brought to bear by the health authorities has been
largely from a sanitary standpoint. About 1908-1909 a milk
ordinance was drawn up, which required the pasteurization
of milk or the tuberculin testing of all cows producing the
milk. The tuberculin test was not or could not be enforced,
which made pasteurization obligatory, Imasmuch as this part
of the ordinance could be enforced. Chicago was the first city
of any size in America to demand pasteurization of market
milk.

No systematic sanitary inspection of farms was established
until 1910. In 1909 the state legislature passed a law provid-
ing that in determining the quality of milk nothing should
be taken into consideration except the result of the analysis
of the milk in the can, which no doubt was a blow to farm in-
spection. This law was repealed in 1915. The Commissioner
of Health hag general supervision of the milk inspection.
There is also a Chief Food Inspector working under the direc-
tion of the Commissioner of Health, and he in turn has super-
visors to carry out his orders.

The entire dairy district is divided into fifteen inspection
districts, each with one inspector. Three inspections per year
per farm are the maximum requirements. If a farmer is
found below standard on the first inspection, a re-inspection

THE MILK SUPPLY OF CHICAGO 181

is made after a reasonable lapse of time. If no improvement
has been made, the milk is excluded until such time as the
rules and regulations are complied with.

In addition there are four ‘‘field platers’’, whose duties
consist in making bacterial tests on pasteurizing equipments
as to efficiency. These men travel from plant to plant con-
tinually.

One of the larger companies maintains its own system of
inspection. One inspector is located at each plant, and visits
each farm delivering milk to that plant at least once every
month. Cooperating with these men, this company has on
its staff a corps of veterinarians, who have direct charge of
the elaborate inspection system of this concern.

The city is also divided into inspection districts, fifteen in
number, with one inspector in each district. These men inspect
the various milk establishments throughout the city and take
chemical and bacterial samples of the various milk products
for analysis at the city laboratories.

The city of Chicago maintains a laboratory for the examina-
tion of milk and cream samples. During the year 1916, this
laboratory examined 44,000 samples of milk and cream chemi-
eally, and 10,000 samples bacteriologically.

On the whole the milk situation has improved in Chicago in
late years from certain points of view. The Health Depart-
ment is continually making strenuous efforts to insure a safe
supply by eliminating as rapidly as possible establishments
that are substandard from a sanitary standpoint.

During the last 15 years, according to Dr. W. A. Evans,
former Health Commissioner, the baby death rate has been
cut in two. The typhoid death rate in 1917 was 1.6 per thous-
and, the lowest of any large city in the world.

THE BACTERIOLOGY OF ICE CREAM
B. W. HAMMER

Within the last few years, the growth of the ice cream in-
dustry has been phenomenally rapid. Instead of being a
product associated with holidays and similar occasions, ice
cream has become a common confection and standard dessert
that is readily available in even the smallest towns. Its manu-
facture has grown from a home, or at least a local affair, to
an industry of immense magnitude with regular shipments
over steam and electric roads to large numbers of dispensing
establishments.

The rapid development of the ice cream business has in-
troduced numerous problems, many of which are essentially
bacteriological in nature. The enormous demands that are
made by the manufacturers on the sweet cream supply,
especially during the warm months when shipping is more
difficult and when the amount of milk produced is ordinarily
decreasing, have necessitated the institution of various ship-
ping procedures. Suitable methods of holding cream have
been required, not only to provide for possible heavy demands
but also to take care of over-stocks during cool weather. Sharp
competition, with the consequent tendency to more efficient
methods and a better product, has likewise necessitated pro-
cedures that inevitably involve bacteriological considerations,
and the public health side has been considered because with
the increase in the consumption, the importance of ice cream
as a means of spreading disease has been realized and an
effort made to decrease the danger from this source.

Tur Numbers or Bacrrerta In Ick Cream

The numbers of bacteria present in ice cream have been
studied by many laboratorics, particularly those associated
with health departments. The cubic centimeter has been

THE BACTERIOLOGY OF ICE CREAM 183

much more frequently used as the basis for determinations
than the gram, since it offers a big advantage in the saving
of time that is possible in the securing of the portion to be
examined. Samples can be conveniently collected in sterile
petri dishes or sterile flasks and can then be slowly and care-
fully melted down and mixed. The thorough mixing of the
sample is very essential as it results in the working out of
the incorporated air that otherwise interferes with the accurate
measuring of the material. Ice cream can be collected from
freezers with a sterile spoon or can be allowed to fall directly
into a petri dish and can be removed from containers of
hardened ice cream by the use of a sterile spoon or a sterile
butter trier. Under ordinary conditions it seems desirable to
discard the ice cream at the surface of containers, and where
a trier is used this can be conveniently done by cutting the
drawn core over the edge of the bottom half of a petri dish
in such a way as to allow as much of the core as is desired
to fall into the dish. A wide range of media has been used
in the plating of ice cream but, since milk derivatives are the
greatest source of the bacteria in ice cream, there seems to
be a very good reason for using the same medium for this
product that is used for milk. The incubation conditions
most commonly used are 37° C. for 48 hours; Ayers & John-
sont found however, that 30° C. for 5 days gave them prac-
tically double the count they secured at 37° C. for 48 hours.

The bacterial counts that have been reported in the liter-
ature vary widely and are in general surprisingly high. Counts
of only a few thousand per ¢. ¢. have been occasionally re-
corded but those running into the hundreds of thousands or
millions are much more common. A count of 8,000,000,000°
per e. c. was secured in Milwaukee. The average of a series
of samples examined in our American cities at the present
time is almost certain to run into the millions per ec. ¢. unless
unusual conditions prevail. Seasonal differences of consider-

i . 22,
LU. 8. Dept. of Agr., Bulletin 563, p ;
2 Bulletin of the Milwaukee Bureau of Economy and Efficiency, No. 13,

p. 35.

184 UNIVERSITY (OF WISCONSIN STUDIES

able importance have been observed. Ayers and Johnson?
working in Washington, D. C. found that 94 samples of ice
cream examined during the summer months showed counts
from 120,000 to 510,000,000 with an average of 37,859,907
p2r ¢. ¢., while 91 samples examined during the winter months
ranged from 13,000 to 114,000,000 and averaged 10,388,222
per ¢. ¢.

The great bulk of the ice cream manufactured in the U. 8.
is vanilla ice cream and accordingly this is the type that
has been dealt with in most of the bacteriological examinations.
The Iowa Agricultural Experiment Station* has found that
the bacterial content of ice cream other than vanilla is es-
sentially the same as that of vanilla and this finding is sub-
siantiated by data published by various laboratories. Water
sherbets, on the contrary, contain comparatively small num-
bers of bacteria; the Iowa Agricultural Experiment Station’
found in 17 samples that the counts ranged from 6 to 7,800
per @. ¢. with no evident relationship between the bacterial
count and the flavor. The small numbers of bacteria in water
sherbets are undoubtedly due to the fact that cream is not
employed in the manufacture, although the acid that is or-
dinarily present in these products may also play a part in
keeping down the count by destroying certain organisms.

Because of the viscosity, the high percentage of solids, and
the large part of the solids made up of fat, the distribution
of the organisms in melted ice cream is perhaps never as
uniform as in a sample of milk. Duplicate determinations
made on unbroken containers of ice cream agree very well
however. In a comparison of 17 duplicates the Iowa Agri-
cultural Experiment Station® found variations from .79 per
cent to 22.73 per cent with an average of 6.75 per cent. On
broken containers from which ice cream was being dipped,
however, much higher variations were found, one of 137.19
per cent being recorded. Ayers & Johnson® have more re-

2U. 8S. Dept. of Agriculture, Bulletin 303.
4 Unpublished data.

5 Bulletin 134, p, 298.

°U. S. Dept. of Agriculture, Bulletin 563.

THE BACTERIOLOGY OF ICE CREAM “185

cently studied the variation between results secured on the
same lot of ice cream and found ‘‘the general variation among
the samples from each gallon of ice cream was from 20
to 30 per cent’’. These investigators consider that the
method used by them gives results ‘‘which indicate that bac-
teria in commercial ice cream are distributed quite evenly
and that an analysis of one sample from a gallon of iee cream
gives results which will hold for any other similar sample
from the same gallon’’. The variation in results secured
on the same lot of ice cream is one of the objections to a
bacterial standard that is advanced by commercial men and
it seems that it is entitled to much study although there is
ample evidence that a bacterial count is a very good index

of the sanitary quality of the material employed and the care
used in the manufacture.

Types oF Bacteria IN Ice CREAM

As would be expected from the wide range of sources of
contamination, practically all of the common types of bacteria
can be found in ice cream, while the prevailing types are
determined by various factors. I£ cream which has been held
under conditions favorable for the development of Bact. lactis
acidi is used, the finished product is practically certain to
contain this organism as the predominating type. In the same
way, other organisms may be present in excessive numbers if
conditions are favorable for their development in any of the
materials used or in the utensils employed. Jee cream with
a low bacterial count is very likely to have the most of the
bacteria of the spore-forming types due to the efficient pas-
teurization and holding.

Avers and Johnson’ studied the types of bacteria that were
present in the ice cream sold at retail in Washington. The
group percentages for 71 summer samples were acid-coagu-
lating 49.82 per cent, acid-forming 20.72 per cent, inert 13.98
per cent, alkali-forming 1.86 per cent, and peptonizing 13.62
per cent, and for 28 winter samples acid-coagulating 30.84 per

7U. S. Dept. of Agriculture, Bulletin 303.

186 UNIVERSITY OF WISCONSIN STUDIES

cent, acid-forming 38.03 per cent, inert 4.81 per cent, alkali-
forming 5.42 per cent and peptonizing 20.90 per cent. In
general these were high count ice creams and would be ex-
pected to show a considerable percentage of acid producing
organisms.

The occasional presence of pathogens in ice cream is evi-
dent from the cases of disease that have been traced to this
product and is to be expected, due to the presence of these
types of organisms in cream.

It ig evident that ordinarily the types of bacteria found in
ice cream are determined practically entirely by the sanitary
quality of the materials used and by the handling given and
that the range in flora is limited only by the extent of varia-
tion that is possible with these.

Tur Sources oF THE BacTEerRIA IN Ick CREAM

The bacteria that are present in ice cream come from two
main sources: (1) the materials used and (2) outside con-
tamination.

(1) The Materials Used

Cream. The cream used in the manufacture of ice cream
is quite likely to carry many bacteria unless pasteurization is
used, since ice cream manufacturers ordinarily buy cream
under conditions such that they have little, if any, control
over the methods of production. Commonly also considerable
difficulty is experienced in securing sufficient sweet cream ;
this is particularly true during the hottest months when the
demands on the manufacturer are heavy. Accordingly, the
ice cream factories in certain sections are sometimes virtually
forced to accept cream with practically no other requirement
than that it be sweet and, in certain cases, the ice cream sold
is ample evidence that this requirement is not rigidly adhered
to. A portion of this cream is shipped without refrigeration
facilities and, although pasteurization before shipment is quite
common, it inevitably arrives at the ice cream factories with
enormous numbers of bacteria.

THE BACTERIOLOGY OF ICE CREAM 187

Under the condition usually prevailing, the modern ice
cream manufacturer finds it very necessary to pasteurize the
cream which he receives. This is necessary because he has no
control over the production and desires to put out a product
containing no living pathogens and also because he wishes
it to have satisfactory keeping qualities, as it is ordinarily
uecessary for him to hold it at least a short time. The pasteur-
ization of cream is usually followed by aging in order that
the ice cream maker may secure the most desirable yield and
texture. The length of the aging depends, under commercial
conditions, on the demand for ice cream and on various other
factors over which the manufacturer has little or no control.
It is usually considered that 48 hours is a sufficiently long
aging period, but this may be reduced due to a heavy demand
or lengthened as a result of cooler weather. The holding of
the cream can be accomplished without an increase in the
bacterial content by the use of proper methods. If the pasteur-
ization is followed by prompt cooling to 32° F. or slightly
below (e. g. until a half inch layer of cream freezes to the
wall of the storage tank) there will be no multiplication of
the bacteria for a considerable period of time. From the re-
sults secured on the storage of cream at 32° F. by the Iowa
Agricultural Experiment Station’ and from data secured by
various investigators on milk held at this temperature, it
seems that even under very favorable conditions cream should
not be held for more than a week, since there are certain
bacteria that will grow in cream at this comparatively low
temperature. For the storage of cream, the use of tempera-
tures which will freeze the cream has been suggested in con-
nection with agitation during the cooling process. Such a pro-
cedure would be expected to prevent the separation of the
solids that occurs when milk or cream is frozen without agi-
tation and may eventually prove widely practical, particularly
when the cream or the ice cream mix is to be homogenized.
The use of temperatures below the freezing point would make
possible the storage of cream for extended periods of time’
without an increase in the bacteria contained.

8 Bull. 134, p. 285.

188 UNIVERSITY OF WISCONSIN STUDIES

Ordinarily cream is one of the very important sources of
the bacteria in ice cream. It is possible that with a very
high grade vream and with some other ingredient of a poor
quality the eream may be over-shadowed as a source of
the bacteria, but this is rarely, if ever, the case under practical
conditions. The extent to which cream may contaminate ice
cream varies from the enormous number of bacteria con-
tributed by a raw cream on the verge of souring to the small
number added by a high grade cream, properly pasteurized
and stored. By the employment of proper pasteurization and
storage, the contamination from the cream can be controlled
and when the added safety to the consumer is considered there
is no justifiable objection to ordinances requiring the pasteuri-
zation of cream used in the manufacture of ice cream.

The homogenization of cream is becoming more and more
common and is playing a very important part in well regu-
lated ice cream manufacture. Ordinarily homogenization in-
creases the bacterial content of cream, as determined by the
plate method, and this is presumably due to a breaking up
of the clumps of bacteria just as is the increase due to the
clarification of milk. The extent of the increase with homo-
genization is very variable and undoubtedly depends on the
numbers and types of the clumps present. When a homo-
genizer is used it should be given careful attention or it may
become an important source of contamination because of the
difficulty of cleaning.

Gelatin. Gelatin may or may not be an important source
of the bacteria in ice cream. Gordon’ has published the bac-
terial contents of 20 samples of gelatin where the results ran
from 200 to 30,000,000 per ec. c and the Iowa Agricultural
Experiment Station?® studied 5 samples and found the counts
varying from 35 to 113,000,000 per gram; from these data it
ix evident that the bacterial content of gelatin is extremely
variable. The heating to which gelatin is subjected in order
to get it into solution before its addition to an ice cream mix
may destroy some of the bacteria, but it does not seem that

"Ice Cream Trade Jr, p. 338, Jan., 1912.
10 Bulletin 134, p. 286.

THE BACTERIOLOGY OF ICE CREAM 189

this can be very important as the best practice prohibits the
use of excessive heat on account of its effect on the solidifying
property of the gelatin and the bacteria present in dry gelatin
must have considerable resistance or the desiccation to which
they have been exposed would have destroyed them. The
bacterial flora of gelatin is, to all appearances, made up largely
of spore formers, and the number of organisms per gram is
presumably closely related to the sanitary quality of the
material from which the gelatin was made. High grade gelatin
can be secured which has a negligible influence on the bacterial
count while certain gelatins may add large numbers of bacteria
to the ice cream mix and are to be avoided in the manufacture
of the highest quality product.

Sugar. If sugar is properly protected its bacterial content
is practically certain to be very low, but it is entirely probable
that where sugar is exposed to dust and dirt it may be of
some little consequence as a means of contaminating an ice
cream mix. The results secured on plates indicate that the
great majority of the bacteria are spore-forming types.

Vanilla. The Iowa Agricultural Experiment Station’ has
examined five samples of vanilla and found the bacterial
content ranging from 200 to 2,300 per c¢. c¢. The alcohol
present in vanilla extract is undoubtedly responsible, in part
at least, for the low bacterial content and moreover this makes
it reasonably certain that vanilla is never an important source
of the bacteria present in ice cream.

Condensed Milk. Condensed milk, usually the bulk con-
densed, is used in enormous quantities in many ice cream
plants. The product at the time of its use has been found by
the Iowa Agricultural Experiment Station’? to contain large
numbers of organisms in certain cases. Excessive numbers
of organisms are in part caused by defective shipping and
holding methods and in some cases perhaps by a poor quality
of material from which the condensed milk is made. Bulk
condensed milk has been found in at least one instance to have

1 Bull. 134, p. 287.
2 Unpublished data.

190 UNIVERSITY OF WISCONSIN STUDIES

been the cause of large numbers of bacteria in the product
of a certain ice cream plant.

(2) Outside Contamination

Air. Air plays a part in the contamination of practically
all dairy products although apparently this contamination is
less serious than it was at one time supposed to be. In an ice
cream plant, as in most dairy establishments, the moisture on
the floors, walls and ceiling must tend to materially reduce
contamination from this source. With wide-open windows and
a dust ladened air blowing in, it is conceivable that air con-
tamination might be of considerable consequence but under
ordinary conditions this is certainly not true.

Utensils. Utensils undoubtedly are as important a source
of the contamination of ice cream as they are of other dairy
products, and always should be given the best of care. The
Towa Agricultural Experiment Station’® determined the con-
tamination from a freezer by adding sterile water to the ma-
chine and then operating the freezer for about the freezing
period, after which the bacterial content of the water was de-
termined. Although the freezer was one that was given more
than ordinary care, the counts in 5 tests were 3,700; 141,500;
8,050 ; 1.195; and 300 per ec. ¢. respectively. The increases per
ec. ec. of an ice cream mix would have been considerably more
than in the case of the water because it was necessary to use
an excessive amount of water to fill the machine since the
water would not hold the air beaten into it and thus failed to
inerease in volume. From these results, it seems evident that
the careful ice cream manufacturer should study his process
with the idea of reducing the contamination from the various
utensils used. Because of its construction the freezer should
be given particular attention; small amounts of water should
not be allowed to stand in it from oue washing until the
next freezing because of the probability of the rapid multipli-
cation of bacteria in this. Steam should be used with great
cere on a freezer, and if the freezing chamber is steamed,

% Unpublished data,

THE BACTERIOLOGY OF ICE CREAM 191

water should be circulated or the freezer slowly cooled in
some other way before the cold brine is circulated.

Employees. The direct contamination of dairy products
from persons is especially undesirable because of the possibility
of the introduction of pathogenic bacteria in this way. The
real danger from employees is shown by several typhoid epi-
demics in which the ice cream causing the infection was con-
taminated by carriers coming in contact with the material
during the manufacturing process. The handling of the cream
after pasteurization should be done under the most careful
conditions, as should also the handling of the materials that
are not heated at all.

CHANGES IN THE Numpers or Bacrerta DuriIna FREEZING,
HaRrDENING, AND HoLpING

The changes in the numbers of bacteria during the freezing,
hardening and holding of the ice cream are of considerable
importance if bacterial standards for this product are to be
employed.

In the modern ice cream freezers, the ice cream mix is sub-
jected to rather violent agitation in addition to having its
temperature brought down below the freezing point of water
and it would be expected that significant changes in the num-
bers of bacteria, as determined by the plate method, might
occur. The lowering of the temperature would be expected
to destroy certain of the bacteria while the agitation would
be expected to break up the clumps of bacteria that might be
present. The Iowa Agricultural Experiment Station’? com-
pared the bacterial content, as determined by the plate method,
of the ice cream mix and the frozen ice cream in 51 cases. In
2 cases (4 per cent) there was no change; in 6 cases (11.8 per
cent) the freezing lowered the count, the decrease varying
from 2 to 31 per cent (Av. 13.0 per cent) while in 43 cases
(84.8 per cent) it increased the count from 2 to 227 per cent
(Ay. 46.3 per cent). A few representative comparisons follow :

144 Unpublished data.

192 UNIVERSITY OF WISCONSIN STUDIES

Trial Bacteria Bacteria in frozen Per cent change
No. in mix ice cream later in numbers
Pere. ec. Perc. ¢.
1 19,100,000 24,750,000 +30
2 2,220,000 2,270,000 +2
3 30,000 33,500 +12
4 485,000 660 ,000 +36
5 27,500 45, 500 +65
6 5,600, 000 4,700,000 —16
7 270,000 285,00) +6

From the results obtained it is evident that freezing com-
monly increases the apparent bacterial content of ice cream
and it seems reasonable to assume that this increase is due to
the breaking up of the clumps of bacteria as a result of the
agitation in the freezer. The lowering of the temperature in
all probability causes the destruction of some of the organisms,
and in those mixes in which there are few or no clumps the
lowering of the temperature may be of greater significance in
determining the change in numbers, as a result of the freezing,
than is the agitation.

The hardening of ice cream that has just come from the
freezer involves a further decrease in temperature and this
would ordinarily be expected to have an influence on the bac-
teria contained. Ice cream usually leaves the freezer at a tem-
perature of 26° to 28° F. and is then reduced to a temperature
usually below 10° F. by using either ice and salt or a refrig-
erated room. The effect of this lowering in temperature has
been studied by the Iowa Agricultural Experiment Station
in 52 comparisons of the bacterial content of the frozen ice
cream before and after hardening. In 1 case (2 per cent)
there was no change in numbers during the hardening; in
45 cases (86.5 per cent) there was a decrease, varying from
2 to 75 per cent (Av. 39.1 per cent), while in 6 cases (11.5
per cent) there was an inerease varying from 7 to 22 per cent
(Av. 13.8 per cent). Illustrative results follow:

1©TInpublished data.

THE BACTERIOLOGY OF ICE CREAM 198

ee A ee
aT eo

rin Bacteria in frozen Bacteria in hardened Per cent change
oO. ice cream ice cream in numbers
Per e. ¢. Per ¢. ¢.
1 2,270,000 1,660,000 —27
2 2,120,000 1,105,000 —48
3 4,700,000 5,200,000 +11
4 33,500 21,000 —37
5 30,500 30,000 eee 4
6 21,000,000 5,320,000 —75
7 268,500 246,000 —s

In general the hardening of frozen ice cream caused a
lowering in the bacterial count, as determined by the plate
method, due presumably to the destructive action of the
lowered temperature. The slight increase in a small percentage
of the cases was in all probability due to experimental error,
as a multiplication of the organisms was very improbable and
care was taken to prevent contamination.

The influence of holding on the bacterial content, of ice
cream is of a great deal of importance from the standpoint
of bacterial standards. Unless definite information regarding
what can be: expected to happen to the bacterial content of
ice cream during storage is available, bacterial standards can-
not justifiably be instituted because holding ice cream un-
doubtedly has some influence on the numbers of bacteria con-
tained. In 1912, the Iowa Agricultural Experiment Station"®
reported data on a number of samples which showed that
in all probability there is a decrease, or else very little change,
in the bacterial content of ice cream during storage and
pointed out certain objections to results that had been pre-
viously presented with the idea of showing an increase in the
numbers of bacteria in ice cream during holding. Esten and
Mason‘? coneluded from their study of 12 samples that when
ice cream is kept frozen for periods of at least a month, there
is no marked inercase or decrease in the bacterial count, as
shown by litmus lactose gelatin plate cultures. More recently

1 Bull. 134. : :
17 Bull, 83, Storrs Agricultural Experiment Station.

13

194 UNIVERSITY OF WISCONSIN STUDIES

the Iowa Agricultural Experiment Station*® has studied 39
samples of ice cream held with ice and salt and 12 samples
held in a commercial hardening room and the results secured
make it still more evident that as long as ice cream is kept
suitably hardened, there is no inerease in the number of
bacteria, but that on the other hand, in many cases, there is
a tendency towards a decrease. The rate and extent of this
decrease is in all probability determined by the types of or-
ganisms present and is due directly to the low temperature
to which the organisms are exposed.

INFLUENCE OF SOFTENING AND REHARDENING IcE CREAM

The softening and rehardening of ice cream occurs fre-
quently in retail establishments when the preduct is not prop-
erly cared for, and accordingly the influence of this procedure
on the bacterial content is of considerable importance. The
Jowa Agricultural Experiment Station?® has studied the in-
fluence of softening and rehardening in a number of instances
and in general the results seem to be quite variable. There
are evidently two influences operative in the softening and
rehardening of ice cream and these act in opposite ways. The
increase in the temperature of the product probably allows
of a certain amount of growth, while, on the other hand, the
subsequent hardening will have the usual destructive effect
on the organisms; whether there will finally be an increase or
a decrease will be determined by whether the multiplication
of the bacteria or the destructive action of the hardening is
the more important.

Manveractvre or Ice Cream with A Low BacrertaL Count

The use of methods that tend to keep down the bacterial
content of ice cream is now widespread among the best com-
mercial ice cream men. They practice pasteurization, the
steaming of containers and equipment, careful protection of
ingredients and various other procedures with the idea of

144 Unpublished data.
Unpublished data.

THE BACTERIOLOGY OF ICE CREAM 195

putting a clean and safe product on the market. The Iowa
Agricultural Experiment Station?® in 1912 attempted the
manufacture of ice cream with a low bacterial count and suec-
ceeded, by using cream that had been properly pasteurized
and well cared for, other ingredients of high quality, and thor-
oughly steamed utensils, containers, and freezer, in producing
ice cream containing only a comparatively few bacteria. The
methods that were employed were selected with particular at-
tention to their practicability and can be used in any properly
equipped factory. The bacterial content of some of the lots
of ice cream is given below.

Trial No, Bacterial content of ice cream

Sites ctaie, decieaeSiata Sie Dente eect e eter e ete ee ee eceeteeseseeees 6,300 DOr C. tu.
IDE ie hie Gee tna hic Bea mB Ak ie BARNES in Se ss a wasn wg aman 10,000 es
Sl BA aren teeta chalice ip kegs ib el dep aided ae sons 4,200 ae

A procedure that has recently been tried out from the
standpoint of its influence on the bacterial count is the pas-
teurization and immediate homogenization of the mix either
shortly before or 24 hours before freezing. Some exception-
ally low count ice cream has been secured in this way, while
in other cases the count has not been quite so satisfactory ;
the variation is, in all probability, due to the variation in
the resistance of the bacteria contained in the cream to the
pasteurization temperatures. The procedure involves the
pasteurization of all the ingredients that go into the ice cream
and not the cream alone. Moreover, it calls for less handling
after the pasteurization than the pasteurization of the cream
and the subsequent preparation of the mix. The procedure is
claimed by commercial men to possess certain advantages from
the standpoint of factory methods, and it seems that its ad-
visability from the standpoint of the wholesomeness of the
product cannot be questioned. The changes secured in the
numbers of bacteria during the procedure are shown in three
trials below.

2 Bull. 134, p. 288.

196 UNIVERSITY OF WISCONSIN STUDIES

Trial I
Material Bacteria per c. c.
CREA: ccoirorccescaclstardnawage den aantesgaietientty Waves Hie nd eacdigibin ini s 9 HLA 720,000
Mix heated but not held.......... cece eee eee eens 156,500
Mix pasteurized 142 degrees F., 20 minuteS....-...+-seeeeeee eran 1,545
Pasteurized mix through homogenizer without pressure......... 2,025
Pasteurized mix through homogenizer, 170 Kg. pressure.......- 4,250
Frozen i€C@ CLEAN 2... cece eee eee ener e ence nee sees tenet 5,900
Hardened ice cream..........c.e cree cere neers ities diestvarete neeoele Soa ae 2,250
Trial IL
Material Bacteria per c. e.
CLOAN. sce dededis ap sa deletetbcinane ae te ae ea eaeKsecebaaeh Ria sone pie suena ME Hie as 5,200,000
Mix before: heating: .e1 a6 cc veneraycecs celeseiong ge Se ws Merisoner crea o's onl nel 1,300,000
Mix pasteurized: os... cc:osaecd 4 24 be Stee tandnes 4 seine Raitceiorets Sameer 170
Pasteurized mix through homogenizer with pressure............. 375
Frozen! ACC CRO AI s4 aa tiered aaheheeanl hers 5 nd aiheaendlgiisie sng aed 7,950
‘Trial IIT
Material
QC ATT asensigoas edb oes-hinisdyond SeueisaeMaA eae uae ASCE IT BE Me Uae See eres

Mix before heating........... 0. ese c eect e ence eee eee tee e rece snes
Mix pasteurized 145 degrees F., 20 minutes...............0.00 eee
Pasteurized mix through homogenizer with pressure.
rozeni ice cream owwasdiiens oy alewame teas 1a oe ie Reet 24 Be

One of the peculiar changes shown in the data is the con-
siderably smaller number of bacteria in the mix than in the
cream. This relationship has been consistently obtained, and
is likely due, in part at least, to the plasmolyzing action of
the sugar. The volume change may play a part, but the very
great reduction encountered in most instances cannot be ex-
plained in that way alone.

Revation or Ick Cream ‘to Pusiic Heavre

The real reason for the concern regarding the bacteriology
of ice cream is that this product is more or less frequently
responsible for human disease. The diseases caused by ice
cream can be divided into two groups: poisoning and infeec-
tious disease.

The eases of poisoning from ive cream are well advertised
by the newspapers and it is very probable that ice cream gets
more than its just share of the blame in this connection. It
seems, moreover, that the most of the ice cream that is the
cause of cases of poisoning is ice cream manufactured in homes
or in small poorly equipped factories. Tee cream mixes made

THE BACTERIOLOGY OF ICE CREAM 197

mp and allowed to stand at a fairly high temperature before
being frozen have been shown to give trouble in certain in-
stances; this is to be expected especially when the material
has been heated and the non-spore-forming acid producers
destroyed, thus leaving the field free to the spore-bearing bac-
teria. The long storage of cream at temperatures around 32°
F. should be prevented because of the well-known chemical
changes that occur in milk and undoubtedly in cream at such
temperatures. The pronounced increase in the amount of
soluble nitrogen is much more likely to result in the develop-
ment of poisonous products than the decomposition of the
lactose which takes place at higher temperatures and which
by the development of acid keeps down the spore-forming
peptonizing bacteria. The ice cream manufacturers have a
real problem in their cream supply, particularly at times of
fairs, holidays, etc., when the storage of cream for consid-
erable periods of time is a practical necessity except in those
exceptional districts where there is an abundant sweet cream
supply. It seems, though, that safety demands a reasonable
limit on the length of time that cream should be stored, even
at such favorable storage temperatures as 32° F.

Epidemics due to ice cream have been noted by various in-
vestigators and have become common enough to no longer
excite any unusual comment. The Iowa Agricultural Experi-
ment Station™? cited some of the references previous to 1912
which give an idea of the extent of the outbreaks up to that
time. Since then, literature on the subject has continued to
appear. The disease most commonly spread through ice cream
is very evidently typhoid fever and some fairlv large epidemics
of this disease have been traced to this source. There is no
reason to doubt the claim that all diseases spread through
milk can be spread through ice cream unless precautions are
taken to prevent it.

Undoubtedly, the most common method of the contamina-
tion of ice cream is by bacillus carriers. Such individuals
have frequently been found to be the cause of epidemics and

4 Bull, 134.

198 UNIVERSITY OF WISCONSIN STUDIES

should certainly be kept out of ice cream plants by every
possible method. It now seems evident also that a negative
Widal is by no means definite proof that a certain individual
is not a carrier. An epidemic in Brooklyn in 1914”? was not
traced to its source, and in 1915 another epidemic in the
same section led to stool examinations of the employees of an
ice cream plant supplying most of the ice cream, with the re-
sult that a man who had been working in the factory in 1914
was found to be a carrier. A typhoid epidemic in California**
was found to have been due to ice cream made by a woman
who had had typhoid 17 years before and who gave only
a partial Widal. This same ice cream also caused poisoning
in the persons consuming it and all but two of the persons
poisoned developed typhoid. A portion of the ingredients
was heated, then cooled and added to whipped cream, after
which the mix was allowed to stand from 6:30 until 1:00
o’cloek without ice, before being made into ice cream.

The presence of organisms producing disease in non-epi-
demic form is very likely to occur unless proper care and
handling are followed.

If pathogenic organisms are present in ice cream, the low
temperatures to which they are subjected during the harden-
ing and holding process cannot be depended on to destroy
them. While there will likely be a falling off in the numbers
of living cells, low temperature cannot be counted on to make
the product safe. At the Iowa Agricultural Experiment
Station,?* ice cream artificially infected with the tubercle
bacillus was found capable of producing tuberculosis in guinea
pigs after one month, which is as long as the tests were made,
this period being considerably longer than ice cream is likely
to be held under practical conditions.

2 Letter from New York City Health Dept.
2 yr, Am, Med. Assn., April 21, 1917.
* Unpublished data.

THE BACTERIOLOGY OF ICE CREAM 199

BacrertaL STANDARDS FOR Icke CREAM

Bacterial standards for ice cream have been set by various
cities but it seems that as yet our information regarding the
bacteriology of ice cream is too meager to admit of the estab-
lishment of fair and safe standards. In at least one instance,
however, the institution of a tentative standard was followed
by a marked decrease in the average bacterial content of the ice
cream offered for sale. Standards which prohibit the sale
of ice cream with ‘‘excessive numbers’’ of bacteria undoubt-
edly are of value and force the manufacturers to give at least
some attention to the quality of the ingredients used and the
care employed in the manufacture. Until better methods of
detecting pathogens in ice cream are available, it does little
good to prohibit the sale of ice cream ‘‘containing disease-
produeing bacteria’’, and it seems more advisable to use nu-
merical standards as the bacterial content gives at least some

index of the quality of the ingredients and the care used in
production.