- JfS
us

Issued October 2, 1912.

U. S. DEPARTMENT OF AGRICULTURE,

BUREAU OF ANIMAL INDUSTRY. BULLETIN 150.

A. D. MELVIN, CHIEF OF BUREAU.

HE BACTERIOLOGY OF CHEDDAR CHEESE.

OUTHERN

f RS!TY OF -CALIFORNIA,
LIBRARY,

*~QS ANGELES, CALIF.
BY

E. G. HASTINGS,

Bacteriologist, Wisconsin Agricultural Experiment Station ,

ALICE C. EVANS,

Bacteriologist, Dairy Division, Bureau of Animal Industry,
AND

E. B. HART,

Chemist, Wisconsin Agricultural Experiment Station.

WASHINGTON:

GOVERNMENT PRINTING OFFICE.
1912

Issued October 2, 1912.

U. S. DEPARTMENT OF AGRICULTURE,

BbREAU OF ANIMAL INDUSTRY. BULLETIN 150.

A. D. MELVIN, CHIEF OH BUREAU.

THE BACTERIOLOGY OF CHEDDAR CHEESE.

E. G. HASTINGS,

Bacteriologist, Wisconsin Agricultural Experiment Station,

ALICE C. EVANS,

Bacteriologist, Dairy Division, Bureau of Animal Industry^
AND

E. B. HART,
Chemist, Wisconsin Agricultural Experiment Station.

WASHINGTON:

GOVERNMENT PRINTING OFFICE.
1912

BUREAU OF ANIMAL INDUSTRY.

Chief: A. D. MELVIN. .

Assistant Chief: A. M. FABBINGTON.

Chief Clerk: CHARLES C. CARBOLL.

Animal Husbandry Division: GEOBGE M. ROMMEL, chief.

Biochemic Division: M. DORSET, chief.

Dairy Division: B. H. RAWL, chief.

Field Inspection Division: R. A. RAMSAY, chief.

Meat Inspection Division: RICE P. STEDDOM, chief.

Pathological Division: JOHN R. MOHLEB, chief.

Quarantine Division: RICHABD W. HICKMAN, chief.

Zoological Division: B. H. RANSOM, chief.

Experiment Station: E. C. SCHBOEDEB, superintendent

Editor: JAMES M. PICKENS.

DAIRY DIVISION.

B. H. RAWL, chief.

HELMEB RABILD, in charge of Dairy Farming Investigations.
S. C. THOMPSON, in charge of Dairy Manufacturing Investigations.
L. A. ROGEBS, in charge or Research Laboratories.
EBNEST KELLY, in charge of Market Milk Investigations.
ROBERT MCADAM, in charge of Renovated Butter Inspection.
2

ADDITIONAL COPIES of this publication
2\. may be procured from the SUPERINTEND-
ENT or DOCUMENTS, Government Printing
Office, Washington, D. C., at 10 cents per copy

LETTER OF TRANSMITTAL.

U. S. DEPARTMENT or AGRICULTURE,

BUREAU OF ANIMAL INDUSTRY,
Washington, D. C., May 24,

SIR: I have the honor to transmit for publication in the bulletin
series of the bureau the accompanying manuscript entitled " The
Bacteriology of Cheddar Cheese," by Messrs. E. G. Hastings and
E. B. Hart, of the Wisconsin Agricultural Experiment Station, and
Miss Alice C. Evans, of the Dairy Division of this bureau.

Cooperative work on the factors concerned in the ripening of
Cheddar cheese has for several years been carried on at Madison,
Wis., between the Dairy Division and the Wisconsin Experiment
Station, and this bulletin is one of a series in which the results of
the work are detailed. Previous experiments have already been de-
scribed in Bulletin 122 of the Bureau of Animal Industry, entitled
" Factors Controlling the Moisture Content of Cheese Curds," and
in Bulletins 7 and 11 of the Wisconsin Station.

In connection with the present study credit is due to Mr. L. D.
Bushnell, now with the Kansas Agricultural Experiment Station,
and Mr. Alfred Larsen, of the North Dakota State Hygiene Labora-
tory, who were formerly bacteriologists in the Dairy Division and
while thus engaged prepared a portion of the data presented in this
bulletin.

Kespectfully, A. D. MELVIN,

Chief of Bureau.

Hon. JAMES WILSON,

Secretary of Agriculture.

CONTENTS.

Page.

Introduction 7

The bacterial flora of milk 8

Differences in composition of cheese 9

The enzym content of cheese 10

Biological problems 10

Bacteriological methods 11

The role of Bacterium lactis acidi in Cheddar cheese 13

Effect of curdling on distribution of bacteria in milk 15

Effect of acid on the expulsion of the whey 18

Effect of acid on the texture of the curd 18

The role of acid in the ripening of cheese 18

The protective action of acid 20

The rate of bacterial growth in cheese 21

Methods of examination 21

Results of the work 22

The enzymic action of lactic bacteria 25

Other groups of bacteria in Cheddar cheese 31

Determinations by milk-dilution and plate-culture methods 31

Determinations by microscopic examination of the cheese 37

The disappearance of bacterial cells in cheese 39

Detailed study of lactic bacilli 40

Acidity produced 41

Forms of lactic acid produced 41

Type of curd produced 42

Fermentation of sugars 43

Form of colonies 43

Thermal death point 44

Morphology 44

Conditions for growth in cheese 45

Solvent effect on milk proteins 47

Coccus forms in Cheddar cheese 48

Chromogenic cocci 50

Liquefying organisms 50

The sequence in development of bacterial groups in Cheddar cheese 51

Summary 52

ILLUSTRATIONS.

PLATE.

Page.

PLATE I. Fig. 1. The action of rennet extract on casein suspended in agar in
the presence and in the absence of acid-forming bacteria. Fig.
2. The effect of acid on cheese 20

TEXT FIGURES.

FIGURE 1. Increase of acidity in raw milk preserved with 3 per cent toluol 29

2. Increase of acidity in heated milk preserved with 3 per cent toluol. 29

3. Typical microscopic fields from cheese No. 54 at different stages in

the ripening process 38

4. Typical microscopic fields from cheese No. 91 at different stages in

the ripening process 39

THE BACTERIOLOGY OF CHEDDAR CHEESE.

INTRODUCTION.

The role of microorganisms in the preparation and ripening of
the various kinds of cheese is a problem that has attracted the at-
tention of bacteriologists since the beginning of the science and since
the establishment of the importance of microorganisms in all de-
composition processes. From the same raw materials cow's milk,
salt, and rennet, which is an extract of the fourth stomach of the
young calf a great number of varieties of cheese are prepared.
These cheeses differ not only in texture, but more especially in flavor
and aroma. From what is known of the importance of microorgan-
isms in the preparation of the products of the fermentation indus-
tries it is evident to one who gives consideration to the question that
undoubtedly microorganisms are as essential in the making of cheese
as they are known to be in the preparation of wine, beer, and other
products.

In the making of wine and beer the desired changes are produced
by a single form of life, the true yeasts, and in the preparation of
any desired type of product attention need only be directed as far
as the causal organism is concerned in order to insure the presence
of the particular variety of yeast that has been found by experience
to form a product that has the desired properties as well as to
insure the absence of harmful forms. In the preparation of still
other products of the fermentation industries not only a single form
of life is essential to produce the desired changes, but two or more
are needed, one of which may work on a certain constituent of the
raw material, the other on another, or the one may serve to make
conditions favorable for the growth of the second, usually by the
preparation of suitable food.

It is evident that as the number of kinds of microorganisms that
are needed to produce the desired changes in any product increases,
the complexity of the problem of demonstrating the role of each
of them is also greatly increased, and it may become most difficult to
prove even whether any particular organism is absolutely essential
in the preparation of the product, to say nothing of demonstrating
its exact role. It is not to be supposed that a single form of life is

8 BACTEKIOLOGY OF CHEDDAR CHEESE.

responsible for the complex chemical changes that occur in the ripen-
ing of any particular variety of cheese, but rather that a considerable
number will be involved.

The fermentation industries include those in which microorganisms
are essential for the production of the desired decomposition changes.
In all such industries the quality of the product will depend on the
raw material and the organisms that are used in the decomposition of
that material. The preparation of cheese has not until recent years
been classed as a fermentation industry, although it is one in which
microorganisms play a dominant role. Here, as in all other similar
industries, if control of the quality of the product is to be obtained,
knowledge must be gained concerning the essential biological agents.
In this paper are presented a summary of the present knowledge of
the bacteriology of Cheddar cheese and the results of a detailed study
of a number of cheeses.

THE BACTERIAL FLORA OF MILK.

In the case of many of the products of the fermentation industries
the essential microorganisms are contained in the raw materials, and
the development of the methods of preparation of the products have
been wholly empirical. Especially is this true of certain kinds of
cheese, the methods of manufacture of which are in some cases cen-
turies old. Even in the case of cheese to which at some stage in
manufacture material containing certain microorganisms is added the
methods of manufacture antedate the science of bacteriology.

The sources from which microorganisms enter the milk are much
the same in all parts of the world. Any natural source, such as the
interior of the udder, the dust from the animal, or even the utensils,
will always furnish certain definite types of microorganisms. By
this is not meant that the flora of milk will always include these
definite types and no others, but that certain forms will always be
present. That this is true is shown by the fact that a sample of
ordinary market milk, if stored at temperatures ranging from 60
to 90 F., will undergo a definite sequence of decomposition changes
with almost unerring certainty. In this sequence three main groups
of microorganisms are prominent; first, the acid-forming bacteria
that change the milk into an acid, semisolid mass, favorable for the
development of the mold that is characteristic of milk Oidium lactis.
This mold, by its gradual destruction of the acid and the establish-
ment of an alkaline reaction, makes it possible for the putrefactive
organisms to develop. When the environment is changed this
sequence of microbial life in the milk is also changed. For example,
milk may be kept at such low temperatures that the acid-forming
bacteria can not grow and the putrefactive bacteria appear first.

DIFFERENCES IN COMPOSITION OF CHEESE. 9

It is thus proper to speak of a characteristic milk flora. By some
it is believed that this flora is so constant that fermentations that
depart from the normal are to be looked upon not as due to the in-
troduction of other forms of microorganisms, but most often to
changes in the properties of the organisms or to changes in environ-
ment that favor the particular organisms which produce the abnor-
mal fermentation. Slight changes in the composition of the milk
may also have a determining influence.

As has been stated, the microorganisms necessary for the prepara-
tion of most kinds of cheese are contained in the milk. Whether one
form or the other is to develop in the cheese depends upon the en-
vironment established by the cheese maker. The manufacture of
cheese is thus a problem in the ecology of microorganisms.

DIFFERENCES IN COMPOSITION OF CHEESE.

The differences due to the methods of manufacture result in differ-
ences in composition of the cheese at the time it begins to undergo
the ripening process, which is to determine whether the cheese is to
be classed as one kind or another. The differences in composition
of the fresh cheese are very largely in the amount of moisture pres-
ent. Since the water that is left in the cheese is whey, carrying in
solution sugar and other constituents of milk, the effect of differ-
ences in moisture is not only in influencing the texture, but also the
composition of the cheese. The difference in moisture content
between Cheddar and Camembert cheese is approximately 25 per
cent. If it is supposed that the moisture of the cheese will have
about the same sugar content as milk, this means that the sugar con-
tent of the Camembert cheese will be about 1 per cent greater than
Cheddar; and since the sugar is all fermented, differences in sugar
content ultimately result in differences in acidity.

One of the basal differences between Cheddar and Swiss cheese is
that during the making of the first that is, the operations before
pressing a large amount of acid is formed in the curd, while in the
case of Swiss no such acid production occurs. Van Slyke and Hart, 1
Boekhout and Ott de Vries, 2 and Van Dam 3 have shown that acid is
held chemically by the proteins of cheese, while sugar is not. Thus
if in the making of one cheese acid is formed, and in another not
formed, the result will be a somewhat different composition, even
though the moisture content of the two cheeses is initially the same.

1 Van Slyke, L. L., and Hart, E. B. A study of some of the salts formed by casein and
paracaseln with acids : Their relations to American Cheddar cheese. New York Agricul-
tural Experiment Station Bulletin 214. Geneva, July, 1902. See p. 60.

Boekhout, F. W. J., and Ott de Vries, J. J. Uber den Kasefehler " Kurz " (Kort).
Centralblatt fUr Bakteriologie, Parasitenkunde und Infektionskrankheiten, Abteilung 2,
vol. 24, No. 5/7, pp. 122-129. Jena, Aug. 2, 1909.

Van Dam, W. Uber die Konsistenz der Kasemasse bei Edamkasen. Centralblatt fur
Bakteriologie, Parasitenkunde und Infektionskrankheiten, Abteilung 2, vol. 32, No. 1/2,
pp. 7-40. Jena, Dec. 5. 1911.

50978 Bull. 15012 2

10 BACTERIOLOGY OF CHEDDAR CHEESE.

Another difference in composition of cheese is caused by variations
in the amount of salt and in the time at which it is added. This dif-
ference is an important one in the case of Cheddar and Swiss cheese.
These differences in the initial composition are sufficient to exert a
great influence on the types of life that are to grow in and on the
cheese.

THE ENZYM CONTENT OF CHEESE.

The role of the inherent enzyms of milk and those of the rennet
extract in the ripening of cheese have been emphasized by the work
of Babcock and Russell and of Von Freudenreich and Jensen. There
remains no doubt concerning the importance of certain enzyms, es-
pecially those of rennet extract, in the ripening of cheese. The
enzymic content of all cheese must be much the same qualitatively,
since they are made from the same raw materials. Again, it is dif-
ficult to see how one enzym can be active in one cheese and not in
other kinds, since the conditions are quite similar as regards reaction
and other factors. Accepting the theory of the specificity of enzyms,
it must be admitted that the products of the activity -of any enzym
will be the same in kind no matter what the conditions under which
it may be working. Hence it does not seem that the enzyms of milk
or rennet can be factors determining the kind of cheese to be made
by any method from the ordinary raw materials, but that the deter-
mining factors must be biological.

BIOLOGICAL PROBLEMS.

The methods of manufacture of cheese result in an environment
that causes certain of the organisms constantly present in milk to
develop in a definite sequence. The empirical methods of the cheese-
maker result in a cheese that approximates, if it is not identical with,
the normal of its kind.

Each of the great commercial varieties of cheese thus presents a
distinct problem for the bacteriologist, who should be able to demon-
strate the constant presence of the essential groups of bacteria. It
can not be expected here any more than in most decomposition proc-
esses that a single specific organism will be concerned, as in the
causation of disease, but rather that groups of organisms will be
present, the basis of grouping being largely biochemical. Neither
can it be expected that one group will grow for a period, then dis-
appear and be followed by a second, but rather that the sequence of
development will be confused.

The work of Thorn on Camembert cheese furnishes an excellent
illustration. It has been shown that a definite balance between the
various forms of life concerned is essential for the normal ripening

BACTERIOLOGICAL METHODS. 11

of this cheese. If through methods of manufacture or through
conditions of ripening this balance is destroyed, the cheese will not
be typical.

The biological factors concerned in the ripening of Camembert
cheese are the acid-forming bacteria, the Camembert mold (Penicil-
lium camemberti) , or its white form (Penicillium camemberti var.
rogeri) ; Oidium lactis; and bacteria which together with Oidium
lactis form a reddish slime on the surface of the cheese. If condi-
tions favor the growth of Oidium lactis at the expense of the penicil-
lium, the cheese will not be normal; if the mold is too luxuriant,
texture is obtained but flavor is not. It is thus evident in the case of
Camembert cheese too wide variations in the balance between the
different forms of life can not occur if a cheese normal in texture
and flavor is to be obtained. A large amount of work has been done
on Camembert cheese and the necessity for the presence of the dif-
ferent forms established, but their exact role has not been shown and
never can be until the chemist devises means of following in detail
the complex chemical changes.

It is not only necessary that the constant presence of any organism
in cheese be demonstrated in order to prove its importance in the
ripening, but it must also be present in sufficient numbers so that
it is certain that growth has taken place in the cheese. The simple
presence of any form is no evidence of its activity. Such an error
was made by Duclaux, one of the first bacteriologists to occupy him-
self with cheese problems. Since there are such a multitude of forms
of bacteria in milk, and hence in cheese, any particular method of
examination of the cheese is likely to bring some one group of
organisms to the front. The methods used by Duclaux in his exami-:
nation of Cantal cheese were fitted to favor the development of the
spore-bearing, liquefying bacteria. Their constant presence, together
with the fact that they produced in pure culture in milk compounds
which he had already demonstrated in the ripe cheese, led him to
consider them an important factor in the ripening of this cheese. It
has been demonstrated since that the conditions in hard cheese do
not permit this class of bacteria to develop.

BACTERIOLOGICAL METHODS.

The modern bacteriological methods are not such as will demon-
strate the presence of all kinds of bacteria in milk, cheese, or any
other substance in which a considerable number of kinds of bacteria
are present, even though all may find conditions that will permit of
growth on the medium employed. It is usually easy to demonstrate
the presence of the form that occurs in greatest numbers, since in
the more lightly inoculated culture plates this form will be present

12 BACTERIOLOGY OF CHEDDAR CHEESE.

in pure culture or else so freed from competition with the other
forms that its growth will not be prevented. For those forms present
in less numbers this condition does not obtain, and usually special
methods must be employed to demonstrate their presence, such as
the use of differentiating media or enrichment cultures. Anyone
who has much experience in plating milk on lactose media has noted
that in certain cases the plates thickly seeded show colonies of but
a single organism, usually the ordinary lactic organism Bacterium
lactis acidi, while on the more thinly seeded plates from the same
sample other forms may appear. In the thickly seeded plates the
lactic organisms, on account of favorable conditions, have grown
most rapidly and by the acid produced have prevented the develop-
ment of other forms, while in the more thinly seeded plates the
colonies may be separated by such a distance that the products of one
colony do not reach the others, each colony then grows as though
it were the only colony on the plate. Hence forms appear on thinly
seeded plates that do not on those crowded with colonies. If the
organism that finds most favorable conditions greatly exceeds in
numbers some other form the latter may never appear on the cul-
ture or be so inconstant as not to attract the attention of the bac-
teriologist. A ratio of 1,000 to 1 or even 500 to 1 may prevent the
detection, by use of the ordinary plate-culture methods, of the
organisms present in smaller numbers. This statement may serve
to explain the results obtained by previous investigators of Cheddar
cheese.

Before 1896 no consecutive bacteriological examinations had been
made of a ripening Cheddar cheese. The first detailed work on this
variety of cheese with which the writers are acquainted is that of
Russell. 1 This work was largely concerned with a quantitative exam-
ination of the cheese during the ripening process by means of gelatin
plates. It established the fact that acid-forming organisms make up
99 per cent and over of the bacteria thus determined. It may be
inferred that the acid-forming bacteria belonged to the Bacterium
lactis acidi group. The work of Lloyd on English Cheddar cheese
led to similar results. The most extensive work on the bacterial
flora of Cheddar cheese is that of Harding and Prucha, 2 who iso-
lated the different types of organisms appearing on cultures made
from 9 normal Cheddar cheeses during the entire period of ripening.
The more than 300 cultures thus isolated were studied in detail and
reduced to 33 groups. Of these 33 groups 4 belonged to the Bacte-
rium lactis acidi group, the only one which was always found, and
it practically always included over 99 per cent of the total germ

1 Russell, II. L. The rise and fall of bacteria In Cheddar cheese. Wisconsin Agricul-
tural Experiment Station, Thirteenth Annual Report, pp. 95-111. Madison, 1896.

2 Harding, II. A., and Prucha, M. J. The bacterial flora of Cheddar cheese. New
York Agricultural Experiment Station, Technical Bulletin 8. Geneva, Dec., 1908.

THE ROLE OF BACTERIUM LACTIS ACIDI. 13

content. The 10 other groups which these investigators classed as
important on account of frequency of occurrence were not found in
all of the cheeses, and it may be inferred that they do not represent
essential factors in the ripening of Cheddar cheese. In speaking of
one cheese the authors say :

The results from this cheese accord with the idea that aside from the lactic
group there is no single group or at least no single species of bacteria abso-
lutely essential to the ripening process.

The work of previous investigators has shown that the liquefying,
the gas-forming, and the 4nert bacteria are not essential factors in
the ripening of Cheddar cheese, since all or any one of these groups
may be absent and yet the cheese may ripen in a normal manner.
It is true that all of these groups are usually represented in Cheddar
cheese, since they are present in milk, but the numbers are small,
and in the case of the liquefying organisms there is no evidence that
growth ever occurs during the ripening process.

Yeasts can not be classed as an important factor since they may be
absent from a normal cheese.

The work of previous investigators may be summarized in the
statement that the group of bacteria represented by Bacterium lactis
acidi is the only one that up to the present has been shown to be of
constant occurrence in Cheddar cheese. This fact- together with the
enormous numbers, amounting to millions and at times over a billion
per gram of the fresh cheese, leaves no doubt of the importance of
this group of organisms in Cheddar cheese, and undoubtedly in all
cheese that undergoes a ripening process.

THE ROLE OF BACTERIUM LACTIS ACIDI IN CHEDDAR CHEESE.

The empirical methods of the Cheddar cheesemaker demand a milk
so far advanced in the acid fermentation that during the few hours
between time of curdling and placing the curd in the press acid de-
velopment is rapid. The increasing acidity, as has long been known,
favors the curdling of the milk by the rennet. Indeed the tests most
frequently used in cheesemaking to determine the " ripeness " or
acidity of the milk are those in which the time of curdling is noted,
when a definite amount of rennet is added to milk at a definite tem-
perature (tests of Monrad and Marschall). The Cheddar maker
desires milk that has just passed through the " period of incubation."
By this expression is meant the time during which no apparent in-
crease in acidity results from the growth of acid-forming bacteria.
This has been supposed to be due to the fact that no acid was formed
by the rapidly growing organisms. More recently it has been pointed
out by Kahn * that this is not the true explanation but that until the

1 Rahn, Otto. The fermenting capacity of the average individual cell (Bacterium
lactis acidi). Science, new series, vol. 33, No. 849, pp. 539-540. New York, Apr. 7, 1911.

BACTEKIOLOGY OF CHEDDAE CHEESE,

bacteria have increased to a great extent the amount of acid formed
is so small as to escape detection. Whatever the explanation, it is
true that the acid-forming bacteria may increase until there are
millions per cubic centimeter and yet the acidity show no change
whatever, as has been shown by Koning 1 and by Burri and
Kiirsteiner. 2

At last the acidity begins to develop with increasing rapidity until
it reaches a point where it begins to exert an inhibitive effect upon the
growth of the organism. In Table 1 are given some data illustrating
the rate of acid development in milk kept at 95 F. It will be noted
in both samples that the initial rate of increase in acid is small, that
it reaches a maximum that may be over 0.15 per cent per hour and
then declines. Both samples of milk were slightly too acid for
cheesemaking.

TABLE 1. Rate of development of acid in milk kept at 95 F.

Hours.

Sample 1:
Acidity

Per cent.
0.24

Per cent.
0.30
.06

.24
.04

Per cent.
0.40
.10

.34
.10

Per cent.
0.47
.07

.16

Per cent.
0.71
.12

.74
.12

Per cent.
1.00
.015

.99
.012

Increase per hour .>

Sample 2:
Acidity

.20

Increase per hour

This rapid increase in acidity is the result of the growth of the
acid- forming bacteria in the favorable environment. In Table 2 are
given the results of the bacteriological examination of two samples of
milk kept at 95 F. for a number of hours. It. will be noted that the
increase in numbers of bacteria, like that of the acidity, goes on with
increasing rapidity until a maximum is reached, after which the
growth is less and less rapid.

TABLE 2. Increase of bacteria per cubic centimeter in milk kept at .9,7 F.
[Number of bacteria expressed in millions.]

Hours.

Sample 1

231

927

1,064

1,167

Sample 2

157

826

1,545

1,982

1 Koning, C. J. Der Silurojrrad dor Milch. [Pharmaceutisch Weekblnd, 1004] Milch-
wirtshaftliches Zentralblatt, vol. 1, No. 7, pp. 289-305, July; No. 8, pp. 337~356,
AiiKust. Leipzig, 1905. See p. 294.

- Burrl, R., nnd Kiirsteiner, J. UntprsuchunRon iibor dio Relfung der Kiisereimilch.
Landwlrtschaftliches Jabrbuch der Schweiz, vol. 24, pp. 437-4G6. Bern, 1910.

EFFECT OF CURDLING.

It is essential that the acid development should be rapid during the
time between the curdling of the milk and pressing the curd. The
modern Cheddar maker insures this by the addition of large numbers
of acid-forming bacteria in the form of a starter.

EFFECT OF CURDLING ON DISTRIBUTION OF BACTERIA IN MILK.

The solid bodies present in the milk will be held by the curd in the
same manner as the formation of aluminum or ferric hydrate in water
enmeshes the solid bodies presentj or the coagulating of albumen in
a solution removes turbidity therefrom, as in the clearing of bac-
teriological media and of wine.

In order to illustrate the effect of curdling on the distribution of
bacteria several samples of milk were subjected to a quantitative ex-
amination. Rennet solution was then added and the curd cut. As
soon as possible a sample of the whey was likewise examined. In
Table 3 are given the data obtained from a number of such examina-
tions. It will be noted that in all cases a unit volume of the whey
contained less bacteria than the milk before curdling. From the
average figures of the determinations approximately 77 per cent of
the bacteria were retained in the curd in the trials made in the
laboratory.

TABLE 3. Number of bacteria per cubic, centimeter in milk and in whey im-
mediately after curdling Laboratory experiments.

[Number of bacteria expressed in thousands.]

Trial No.

Milk...

128,000
34,000

11,000
2,900

2,050
730

155,000
75fi

23,000
773

59,000
776

Whey

A number of similar examinations were made under practical con-
ditions in the cheese room, the sample of milk being taken from the
cheese vat and the whey after cutting the curd in the usual manner.
The data are given in Table 4. It will be noted that the results are
similar to those obtained in the laboratory. Approximately 73 per
cent of the bacteria in the milk were retained in the curd.

TABLE 4. Number of bacteria pet- cubic centimeter in milk <in<l in irlicy im-
mediately after curdling Samples from the cheese vat.

[Number of bacteria expressed in thousands.]

Trial No.

Milk

5,600

6,400

5,430

4,000

6,500

Whey

3,000

1,290

2,400

210

4CO

BACTERIOLOGY OF CHEDDAR CHEESE.

The result of curdling and the shrinking of the curd is that the
acid-forming bacteria of the milk are concentrated in the curd, which
soon after cutting occupies but a fraction of the volume of the origi-
nal milk. This concentration, together with the favorable environ-
ment, results in a rapid formation of acid in the curd.

This condition should result in a more rapid increase of acid in a
whey in which the curd is allowed to remain than in a portion of
the same whey removed from the curd. As the curd shrinks the
expelled whey should bring with it a portion of the acid that has
been formed in the curd; this, together with osmotic action, should
increase the acidity of the whey in contact with the curd.

In order to demonstrate this a sample of milk was curdled, the
curd cut, and as soon as possible a portion of the whey removed to
a separate vessel. Acidity determinations were made at intervals.
The results are given in Table 5. It will be noted that in every case
the acidity of the whey in contact with the curd increased much
more rapidly than that of the whey not in contact with the curd.

TABLE 5. Development of acid in ivhey alone and in ichey in contact icith curd.

Hours.

Sample 1 :
Whey

Per cent.
0.09

Per cent.
0.10

Per cent.
0.13

Per cent.
0.16

Per cent.
0.24

Per cent.
0.36

\V hey and curd

.09

.10

.16

.17

.32

.47

.54

Sample 2:
Whey

.14

.20

.30

.35

.49

"Whey and curd

.14

.27

.48

.53

.68

Sample 3:
Whey

.10

.12

.22

.33

Whey and curd

.10

.12

.16

.32

.49

Sample 4:
Whey

.09

.10

.11

.13

.14

.33

Whey and curd

.09

.10

.13

.18

.20

.42

It was thought that if a sample of milk was curdled by rennet and
the curd cut and allowed to settle to the bottom of a deep container,
the whey at different levels should show varying degrees of acidity
if the container were so protected that convection currents did not
tend to mix the liquid. In other words, the acid whey expelled would
tend to accumulate in the lower portion of the container.

In order to test this, deep beakers were filled with milk, the milk
curdled with rennet, and the curd cut and allowed to settle. The
beakers were kept in a thermostat at 95 F. ; thus no convection cur-
rents were present, since care was taken to heat the milk to the same
temperature before curdling. The samples of whey were removed
in such a manner as not to mix the different layers. In Table 6 the
results of a number of trials are given. It will be seen that the
acidity of the bottom layers increases much faster than that of the
upper layers, the difference being in some cases 0.4 per cent.

ACIDITY OF MILK.

TABLE 6. Acidity of whey at the top and bottom of a vessel containing whey

and curd.

Hours.

Sample 1:
Top

Per cent.
0.09

Per cent.
0.11

Per cent.
0.12

Per cent.
0.16

Per cent.
0.27

Per cent.
0.40

Bottom

.09

.10

.13

.22

.49

.67

.68

Sample 2:
Top

.14

.21

.32

.36

.56

Bottom

.14

.34

.64

.71

.80

Sample 3:
Top

.08

.09

.12

.18

.24

Bottom

.08

.09

.11

.15

.34

.45

Sample 4:
Top

.09

.10

.13

.14

.17

.25

Bottom

.09

.10

.14

.22

.24

.59

It is thus shown in a number of ways that the location of acid
development is in the curd rather than in the whey.

It has been shown that paracasein will absorb and probably com-
bine with acids. If this is so, a portion of the acid formed in the
curd should be retained in loose chemical combination and the acidity
of a sample of milk should increase more rapidly than that of the
whey from the same milk, even though the w T hey is in contact with
the curd. The data given in Table 7 show this to be true. The
figures represent the increase in acid at the end of each period over
the initial acidity of the milk or whey and curd. This leaves no
doubt that a portion of the acid is retained by the curd, and is proof
of an earlier statement, that a cheese in which acid is developed dur-
ing the process of making will have a different acidity from one with
an equal content of moisture, but in which no acid is formed during
the making.

TABLE 7. Increase in acidity of milk and of whey in contact with curd.

Hours.

2 '

Sample 1:
Milk

Per cent.
0.19

Per cent.
0.34

Per cent.
0.64

Per cent.
0.73

Per cent.

Percent.

.13

.32

.38

.52

Sample 2:
Milk .

.00

.01

.09

.32

0.55

0.74

Whey and curd

.00

.01

.02

.05

.18

.26

Sample 3:
Milk

.04

.21

.44

.69

Whey and curd

.05

.11

.16

.28

Sample 4:
Milk

.03

.15

.43

.63

.03

.04

.16

.25

50978 Bull. 15012 -3

18 BACTERIOLOGY OF CHEDDAR CHEESE.

EFFECT OF ACID ON EXPULSION OF THE WHEY.

It has been shown by Sammis, Suzuki, and Laabs 1 that the expul-
sion of whey from the curd is directly proportional to the amount
of acidity present; at least, in the case of percentages of acidity that
are met with in normal cheese curds. The growth of lactic-acid
bacteria in the curd and the consequent development of acidity are
necessary to secure a curd with the proper moisture content.

EFFECT OF ACID ON THE TEXTURE OF THE CURD.

The curd from milk in which no growth of acid-forming bacteria
has taken place shows but a slight tendency to mat or for the pieces
of curd to fuse. If, however, acid is formed in the pieces of curd,
they undergo such a change in texture that as soon as they are allowed
to settle to the bottom of the vat they soon unite to form a single
mass of curd. In the making of cheese by the Cheddar process it is
essential that this matting take place.

The change in texture which the curd undergoes is due to the action
of the acid on the paracasein, forming a substance that, when wanned,
can be drawn into threads. The basis of the " hot iron " test as used
to determine the time to draw the whey is the formation of this
compound by the acid.

At the time the curd is placed in the press there are numerous
spaces between the curd particles. It is essential that the curd be
so plastic that, under the influence of the pressure, the particles
undergo perfect fusion, so that the entire cheese is one mass, per-
fectly free from irregular-shaped mechanical holes. Such a fusion
does not occur in the absence of acid formation in the curd.

THE ROLE OF ACID IN THE RIPENING OF CHEESE.

In the ripening process proper the acid resulting from the fer-
mentation of the sugar by the organisms of the Bacterium lactis
acidi group has an important role. As has been shown by numerous
investigators, the sugar in the cheese is all fermented within a few
days. It was shown by Babcock and Kussell, 2 also by Jensen, 8 and
by Van Slyke, Harding, and Hart 4 that rennet extract not only has
a curdling effect but has a digestive action in the presence of an

1 Sammis, J. L. ; Suzuki, S. K., and Laabs, F. W. Factors controlling the moisture
content of cheese curds. TJ. S. Department of Agriculture, Bureau of Animal Industry,
Bulletin 122. Washington, 1910.

a Babcock, S. M. ; Russell, II. L. ; and Vivian, A. Influence of rennet on cheese ripen-
ing. Wisconsin Agricultural Experiment Station, Seventeenth Annual Report, pp. 102-
122. Madison, 1900.

8 Jensen, Orla. Studlen tiber die Enzyme 1m Kase. Landwlrtschaftllches Jahrbuch
der Schwelz, vol. 14, pp. 197-233. Berne, 1900.

4 Van Slyke, L. L. ; Harding, II. A. ; and Hart, E. B. Rennet-enzyme ns a factor In
cheese ripening. New York Agricultural Experiment Station, Bulletin 233. Geneva,
June, 1903.

THE ROLE OF ACID IN RIPENING OF CHEESE.

activating acid such as is present in the cheese. The rapid proteoly-
sis occurring during the first part of the ripening process is largely
due to the action of the pepsin.

Variations in the amount of rennet extract added to the milk result
in differences in the rate of ripening, as has been shown by numerous
investigators. This can be explained only through the variations in
the pepsin content of the cheese. The action of rennet extract in
the presence and absence of lactic organisms is demonstrated in
Plate I, figure 1. A 4 per cent solution of agar was added to sterile
milk in the proportion of 1 to 1. A portion of the milk agar
was heavily inoculated with a lactic organism and the plates in-
cubated for 24 hours; the other portion was not inoculated. At the
end of the period of incubation strips of filter paper were moistened
with rennet extract and placed on the plates, which were then in-
cubated at 37 C. for one hour. The photograph was taken at
the end of the period. It will be noted that in the presence of the
lactic organisms the casein has been rendered soluble by the rennet
extract, while in the absence of the organisms no such marked
digestion has occurred. The digestion of the casein has resulted in
the destruction of the opacity of the milk agar so that a number
placed beneath the dish can be read, while in the case of the milk
agar from which the lactic organisms were absent no trace of a
similar number can be distinguished. In the milk agar conditions
are very similar to those obtaining in cheese, the activating acid being
of like origin.

It would thus seem that if a cheese was made from milk that con-
tained but few acid-forming organisms the rate of ripening would be
delayed. In order to test this hypothesis, a cheese was made from
very clean milk, containing scarcely any acid-producing organisms,
and no starter was added. No acid whatever was formed during the
making process. The milk after standing 24 hours at 20 C. had
an acidity of but 0.19 per cent. In order to insure proper curdling,
the acidity of the milk was raised to 0.25 per cent by the addition
of hydrochloric acid. In order to follow the rate of acid formation,
determinations of the sugar present in the cheese were made at
intervals. The results are given in Table 8.

TABLE 8. Sugar content of cheese made from milk containing few lactic

bacteria.

Time.

Sugar con-
tent.

Acidity.

Time.

Sugar con-
tent.

Acidity.

Days.

Per cent.

Percent.

Days.

Per cent.

1.51

.25

1.45

.27

1.49

.27

.94

.45

1.48

.27

.00

1.13

20 BACTERIOLOGY OP CHEDDAR CHEESE.

The sugar disappears from a normal cheese in 3 to 5 days, while
on the twenty-second day over one-half of the sugar remained in
the experimental cheese. The maximum bacterial content, as meas-
ured by the ordinary plate cultures., was not attained until after
the sixth week. The rate of ripening, as measured by the change in
texture and development of flavor, was correspondingly slow. At
three months the cheese still showed a spongy texture and scarcely
any cheese flavor.

THE PROTECTIVE ACTION OF ACID.

The putrefactive bacteria of the groups that are constantly present
in milk, and hence in cheese, are unable to grow on account of the
acid reaction which is maintained during the entire period of ripen-
ing. This protective action of acid was first demonstrated at the
Wisconsin Experiment Station by Babcock and Russell, 1 who re-
moved the sugar from curd by washing in water. The cheese de-
veloped most undesirable odors and tastes, and bacteriological exami-
nations showed liquefying bacteria to be numerous. If lactose,
glucose, or cane sugar were added to the washed curd, the ripening
process was much more nearly normal because the acid reaction was
thereby restored, due to the fermentation of the added sugar, and
the putrefactive organisms were inhibited as in a normal cheese.
In the experimental work on flavor development in Cheddar cheese,'
a number of cheeses were prepared from curd from which the sugar
was removed by washing. In Plate I, figure 2, a normal and a
washed-curd cheese are illustrated. The washed-curd cheese was
devoid of texture, being a soft, plastic mass and having no resem-
blance to cheese in odor and taste. On the right of the picture is a
cheese made from the same washed curd to which acid had been
added, with the result that the firmness was restored.

The role of organisms of the Bacterium lactis acidi group in
Cheddar cheese may be summarized as follows:

1. They favor the curdling process.

2. They favor the expulsion of the whey.

3. They permit of the fusing of the curd particles.

4. They activate the pepsin of the rennet extract.

5. They have a protective action against the putrefactive bac-

teria.

It is certain that this group is an essential factor in the ripening
of Cheddar cheese. Their period of growth has been believed to be
short, since it has not been supposed they can continue to develop
after the disappearance of the sugar. It has been shown by numer-

1 Babcock, S. M., Russell, H. L., Vivian, A., and Hastings, E. G. Influence of sugar on
the nature of the fermentations occurring In milk and cheese. Wisconsin Agricultural
Experiment Station, Eighteenth Annual Report, pp. 162-176. Madison, 1901.

BUL. 150, BURCAU OF ANIMAL INDUSTRY, U. S. DEPT. OF AGRICULTURE.

PLATE I.

FIG. 1. THE ACTION OF RENNET EXTRACT ON CASEIN SUSPENDED IN AGAR IN THE
PRESENCE AND IN THE ABSENCE OF ACID-FORMING BACTERIA.

The same number (902) was placed beneath both plates at the time the photograph was taken.
The digestion of the casein in the presence of the acid-forming bacteria has rendered the
medium transparent and the number apparent, while in the absence of the acid-forming bac-
teria the digestive action has been almost nil.

FIG. 2. THE EFFECT OF ACID ON CHEESE.

On the left is a cheese made from normal milk by the usual process; in the center a cheese
made from the same milk, the curd being washed free of sugar; on the right a cheese made
from the same washed curd, to which acid had been added. The washed-curd cheese had no
body and was a soft, plastic mass. The addition of acid restored the firmness of the body.

RATE OF BACTEEIAL GKOWTH. 21

ous investigators that the period of maximum numbers is "when the
cheese is a few days old; that thereafter the decline in numbers is
more or less rapid. In some cheeses, as will be shown later, high
numbers may persist for long periods of time. It is not usually
believed that they can exert any marked action in other lines than
those that have been mentioned after growth ceases, although such
a belief may not be well founded. Indeed, some facts would indicate
otherwise.

The ripening process, both in regard to proteolysis and develop-
ment of flavor, continues long after what is supposed to be the period
of bacterial activity. A large amount of data is available as to the
proteolytic changes going on in the ripening cheese, but until recently
no data have been available to show that the compounds containing
no nitrogen were likewise constantly changing. The' content of the
cheese in volatile fatty acids is constantly changing, and especially
the relative amounts of the different acids. To explain these changes
recourse must be had to enzyms, and most probably to those elabo-
rated by the lactic organisms or to other types of bacteria that de-
velop subsequently to what has been called the period of bacterial
activity.-

The data to be presented will show that Ihe period of activity is
not to be measured by determining the ordinary types of bacteria.
The demonstration of continued bacterial growth does not, of course,
eliminate the action of enzyms formed by the Bacterium, lactic acidi
group of organisms that during the early life of the cheese make up
most of the flora.

THE RATE OF BACTERIAL GROWTH IN CHEESE.

In the course of the cooperative work carried on by the Dairy
Division of the Department of Agriculture and the Wisconsin Ex-
periment Station a large number of cheeses have been examined
bacteriologically. Some were examined at frequent intervals during
the making process and during the first portion of the ripening
period; others were examined at less frequent intervals during the
entire period of ripening.

METHODS OF EXAMINATION.

The method of examination used was the grinding with sterile
quartz sand of a sample of the cheese taken from various parts of a
plug and handled under aseptic conditions. Inoculations in vary-
ing dilutions were made from the cheese suspension into lactose-agar
plates. At first the period of incubation of the plates was 48 hours
at 37 C. Later the incubation at 37 was supplemented by a further
incubation at 20. This method of incubation allows the develop-

22 BACTERIOLOGY OF CHEDDAR CHEESE.

merit of the maximum number of organisms and insures the growth
of the colonies to a maximum size, an important point when it is
desired to differentiate the various types present. Gelatin containing
no added sugar was used in a portion of the work; the plate cultures
were then incubated at 18 to 22 C. for 10 days and for even longer
periods when slightly lower temperatures were employed.

RESULTS OF THE WORK.

The data collected by means of the methods that have been used by
other investigators of Cheddar cheese serve simply to confirm their
work and to emphasize the number of organisms of the Bacterium
lactis acidi group that are to be found during the early part of the
ripening period. A portion of the data is presented in Tables 9, 10,
and 11 because it gives a very complete picture of the bacterial de-
velopment during and subsequent to the making of the cheese.

The maximum number of bacteria as measured by the lactose-agar
plate cultures occurs early in the ripening process. In 8 of the 11
cheeses examined, as shown in Tables 9 and 10, the greatest number
of bacteria was found within 48 hours. In the case of the examina-
tions detailed in Table 11, it will be noted that the maximum numbers
of bacteria as determined by lactose-agar plates have been found
much later, namely, at the end of the fourteenth, forty-fifth, and
seventy-seventh days. Thus, in no case was the maximum number
found at the first determination. It is not believed, however, that
these cheeses, selected especially to illustrate certain points to be dis-
cussed later, give a true picture of the initial development of bacteria.
In the case of 9 of the 13 to be mentioned later, the maximum number
of bacteria was found on the first, second, or third examinations.
Harding and Prucha 1 obtained similar results. Seven out of 9
cheeses examined showed the maximum number of bacteria at the
first examination, 1 at the second, and 1 at the fourth.

It is impossible to determine the time at which the growth of any
particular type of organism in cheese ceases. As the fermentation
in the cheese progresses and the accumulation of by-products in-
creases, cell death begins to occur. As long as the process of cell
division is more rapid than death of the cells, an increase in living
bacteria will be shown by the plate cultures. Soon death of the cells
is more rapid than cell division. At this point the decrease in ap-
parent numbers begins, although growth may continue for a much
longer period. It seems probable that the growth of the bacteria of
the Bacterium lactis acidi group continues until the sugar is com-
pletely fermented. In milk the cessation of growth is due to the

1 Harding, H. A., and Prucha, M. J. The bacterial flora of Cheddar cheese. New
York Agricultural Experiment Station, Technical Bulletin 8. Geneva, Dec., 1908.

RATE OF BACTEEIAL GEOWTH.

appearance of free acid, in cheese to the disappearance of an essential
food, a fermentable carbohydrate.

The large numbers of bacteria found during the first days are
striking; the number as given in the tables is far below the actual
number, due to the impossibility of breaking up the colonies in the
tough cheese. It is probable that the germ content of cheese often
amounts to hundreds of billions of living bacteria in each gram of
the moist cheese. From what is known of the number of cells in a
gram of moist bacterial growth, it is certain that at the time the
maximum numbers of the organisms of the Bacterium lactis acidi
group are found, at least 0.1 per cent of the moist cheese consists of
bacteria.

TABLE 9. Numbers of bacteria per gram in Cheddar cheese as determined by
lactose-agar plate cultures.

[Numbers expressed In millions.]

Cheese

ys.

No.

434

490

637

489

438

4.5

174

134

292

442

1.3

971

831

133

221

474

1,657

987

325

407

239

141

475

158

1,538

1,344

987

545

736

476

138

1,311

2,518

616

195

293

728

477

1,925

2,171

1,916

221

598

TABLE 10. Numbers of bacteria per gram in Cheddar cheese as determined by
lactose-agar plate cultures.

[Numbers expressed in millions.]

Curd

Days.

Cheese
No.

Milk.

salt-

12
hours.

time.

580

160

332

586

235

145

165

284

285

104

132

128

114

581

0.5

326

1,048

736

405

684

184

211

290

453

261

228

291

212

582

912

623

709

84S

522

853

369

348

81.4

326

436

193

583

839

965

569

580

1,025

184

401

319

144

504

601

168

BACTERIOLOGY OF CHEDDAR CHEESE.

TABLE 11. Numbers of bacteria per gram in Cheddar cheese as determined by

plate cultures.

[Numbers expressed in millions.]

Number of days.

Cheese No. 1.

Cheese No. 54.

Cheese No. 91.

Cheese No. 92.

Lactose-
agar
culture.

Gelatin
culture.

Lactose-
agar
culture.

Gelatin
culture.

Lartose-
agar
culture.

Gelatin
culture.

Lactose-
agar
culture.

Gelatin
culture.

150

250

150

4 . .

120

400

340

1,400
58

530
1,600

500
35

2.5
2

16
10

270
1,400
1,300

480

100

340

225

32
51

51
40

250

225

36.6

128
74
145
50
18
15
17.5

320
65
142
28.5
23.5
10.5
5.5

250
100
132
145
38.5

84
120
87
96
38
32

100

8.5

113

124

22.5

12
12.5

143

155

165

63
9
12.5

176...

3.5

2.5

5.5

187

7.5

According to the determinations of MacNeal, Latzer, and Kerr, 1
there are about 5,300 billion colon bacilli in a gram of dry growth.
Figuring on a moisture content of 90 per cent, there would be 530
billion in a gram of the moist growth. From determinations made
by weighing out 1 gram of the moist growth of a coccus form from
cheese, making a uniform suspension of this in a known volume of
water, and taking an equal volume of this suspension- and of normal
blood and counting the number of bacteria and red corpuscles we
have obtained an average figure of 1,150 billion cells per gram of the
moist growth. The basis for this method of counting is the fact that
from a known number of red blood cells one can figure the volume
used, since the red cells are practically a constant quantity. The
average volume of the colon organism is 1.13 cubic microns; of the
coccus 0.5236. The agreement in the determinations is thus very close.

It is not to be supposed that, so far as the number of bacteria is
concerned, all cheese will be similar. The results shown in Table 11
emphasize this point ; two of the cheeses, Nos. 1 and 54, showed at no
time over a few million bacteria per gram, while the remaining two
had a consistently high germ content. Such differences may be due
to many causes. The period of maximum numbers is followed by a
decline, rapid in some instances, slow in others. At the time the
cheese is fully ripe millions of living lactic bacteria are usually pres-

1 MacNeal, Ward J. ; Latzer, Lenore L. ; and Kerr, Josephine E. The fecal bacteria of
healthy men. Journal of Infectious Diseases, vol. 6, No. 2, pp. 123-169, Apr. 1; No. 5,
pp. 571-309, Ndv. 26. Chicago, 1909.

THE ENZYMIC ACTION OP LACTIC BACTEEIA. 25

ent in each gram of cheese. Living lactic organisms have been found
by one of the authors in a cheese over 4 years old.

The ripening of the cheese, both in reference to proteolysis and
flavor development, continues long after this group of organisms has
ceased to grow. A large amount of data is available to show that a
constant change in the nitrogenous bodies present is taking place. It
has also been shown by the authors that the content of the cheese in
fixed and volatile acids changes during the ripening process. To
explain these latter changes, recourse must be had to enzyms elab-
orated by the lactic bacteria, or else it must be supposed that other
groups of organisms develop subsequently.

THE ENZYMIC ACTION OF LACTIC BACTERIA.

The work of Buchner, Herzog, and others has shown the presence
of an enzym in certain lactic-acid-producing bacteria that are essen-
tially different from those predominating in cheese. This intracellu-
lar enzym, which can be demonstrated only after the disintegration
of the cell, forms small quantities of lactic acid from sugar. So far
as is known to us, a similar enzym has never been demonstrated in
organisms of the Bacterium lactis acidi group. The growth of these
organisms on all media is so meager that it is very difficult to obtain
a sufficient amount of the growth so that it can be treated by methods
similar to those employed by Buchner and others. An acid-produc-
ing enzym in the lactic bacteria has, however, been demonstrated by
quite different methods. It had been noted that when a sample of
raw or sterilized milk in which varying numbers of lactic bacteria had
been allowed to develop, and to which a preservative, as chloroform
or toluol, had been added, the cells soon disappeared, or at least could
no longer be detected by microscopical examination. It was thought
that if the cells would undergo disintegration after having been
killed by an antiseptic while in an actively growing condition, any
enzyms present should exert their peculiar action as well as though
the cells were mechanically ruptured.

The following experiment was planned : Bottles of fresh raw milk
and of the same milk heated to 97 C. for a short time were in-
oculated with a pure culture of Bacterium lactis acidi. At varying
periods in the development of acid a portion of the milk was re-
moved and preserved with 3 per cent of toluol. In the bottles treated
soon after inoculation a small number of bacteria were present, while
in those to which the preservative was added at a later stage in the
development of acid a much larger amount of bacterial growth was
present. If any enzymic action occurred, the bottles should show a
quantitative difference in the amount of acid formed corresponding
to the amount of bacterial cells present. Raw milk, when preserved
50978 Bull. 15012 4

26 BACTERIOLOGY OF CHEDDAR CHEESE.

with chloroform or toluol, gradually increases in acidity. This in-
crease occurs when the milk contains only the bacteria that come
from the interior of the udder and to which the preservative has been
added as soon as drawn. In a bottle of milk put up in 1898 by Dr.
S. M. Babcock and preserved with an excess of chloroform an acidity
of 0.7 per cent was found in 1910, while the sugar content was as
great as in the fresh milk, being 5 per cent. This increase of acid has
been shown in all samples of milk preserved by the authors. It is
undoubtedly due to the formation of amino acids by the inherent
proteolytic enzyms of the milk.

In the bottles of raw milk in the experiment two acid-forming
factors might be present : First, one forming an acid from the pro-
tein ; second, the enzym of the lactic bacteria acting on the sugar. In
the bottles of heated milk only the latter could be active, since the
degree of heat was sufficient to destroy all the inherent enzyms of
the milk. The milks thus differently treated should show a quantita-
tive difference in increase in acid if the bacterial enzyms were capable
of such action. The first bottle of the raw-milk series contained a
minimum number of bacteria, since the milk was but a few hours old
and drawn under clean conditions. The acidity in this bottle was
that of the fresh milk. Three per cent of a pure lactic organism in
milk was then added to the remainder of the raw milk. The second
bottle was filled immediately after the addition of the culture. The
milk was then incubated and at varying intervals bottles were filled
and toluol added. The first acid determinations were made at once
after adding the toluol to each bottle. It will be noted in Table 13
that the acidity in the case of the raw milk varied from 0.184 to 0.408
per cent. The increasing acidity indicated a great increase in the
numbers of bacteria, which was also shown by microscopical prepa-
rations made from each bottle at the time the preservative was added.

The set of bottles filled with heated milk were treated in the same
manner as the raw milk. The first bottle of this set contained prac-
tically no living bacteria. The acidity in the case of the remainder
ranged from 0.203 to 0.456 per cent.

In order to determine how long the cells persisted in an active con-
dition, tubes of milk were heavily inoculated from the various bottles.
The results are given in Table 12.

RESULTS OF INOCULATION OF STERILE MILK.

TABLE 12. Results of inoculations of sterile milk from the bottles of milk con-
taining 3 per cent toluol.

Bottle
No.

Tube.

Growth from inoculations
made after

Iday.

3 days.

5 days.

2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2

+
+

+
+
+
+

+
+

8.. .

9 .

10
11
12
13
14

The results of the inoculation of sterile milk show that no growth
could have taken place in the bottles after the addition of the toluol,
and that within a short time the lactic organisms were all destroyed.
Any acid formed in the raw milk after the fifth day must have been
due in part, at least, to the inherent proteolytic enzyms of the milk ;
any increase of acid in the heated milk must have been due to the
enzyms set free by the disintegrating cells.

Microscopical preparations were made at intervals from the various
bottles. The smears were stained with a saturated aqueous solution
of methyleiie blue. With this stain the cells can be distinguished
for some time after they can no longer be demonstrated by the Gram-
Weigert stain, which has been used in the examination of smears
direct from cheese (See p. 32.) In raw milk the cells were agglu-
tinated into large clumps. The number in the preparations made
at various intervals became less and less, and after 45 days they had
completely disappeared. As the cells deteriorated they stained more
and more faintly.

In the heated milk the disappearance of the cells was less rapid.
After 74 days some could be distinguished, although their outlines
seemed to be more or less distorted.

In the bottles the following conditions were present : The mass of
cells varied widely; cell growth undoubtedly ceased as soon as the
antiseptic was added and within a few hours the cells were all de-
stroyed, having been killed while in an active condition by an agent
that is supposed to have the minimum effect on the intracellular

BACTERIOLOGY OF CHEDDAR CHEESE.

enzyms. The cells disintegrated more or less rapidly. It would
seem that if any acid- forming enzyms were present in the bacterial
cells they should manifest themselves under these conditions by the
formation of acid. Acid determinations were made with the greatest
precautions possible under the conditions of the experiment. The
results are given in detail in Table 13 :

TABIE 13. Increase of acidity in milk preserved with 3 per cent toluol.

RAW MILK.

When
preserv-

Days.

Sample.

ative
was
added.

121

150

Bottle No. 1:
Vcidity

Per cent.
184

Per cent.
0. 19

Per cent.
234

Per cent.
273

Per cent.
29

Percent.
317

Per cent.
368

Increase..

.006

.05

.089

.106

.133

184

Bottle No. 2:
Acidity

.204

.22

.257

.299

.326

.349

.414

414

Increase

.016

.053

.095

.122

.145

.21

Bottle No. 3:
Acidity

.223

.261

.308

.377

.359

.515

506

Increase

.038

.085

.154

.136

.177

.292

283

Bottle No. 4:
Acidity

266

261

326

345

391

414

492

494

Increase..

005

.06

079

125

.148

226

228

Bottle No. 6:
Acidity

282

332

404

.492

423

644

584

Increase

.03

.122

168

.21

.141

362

302

Bottle No. 6:
Acidity

327

.356

.408

.443

.515

.538

644

676

Increase

.029

.081

.116

.188

.211

317

349

Bottle No. 7:
\cidity

.408

.489

.561

.579

.616

.63

.745

823

Increase

.081

.153

.171

.208

.222

.337

.415

HEATED MILK.

Bottle No. 8:
Acidity....

0.184

0.18

0.175

0.197

0.212

0.243

0.262

267

Increase.. ...

004

009

013

028

059

078

083

Bottle No. 9:
\cidity

203

.206

202

225

258

276

Increase

.003

.001

.022

.055

.067

.073

Bottle No. 10:
Acidity

.256

285

.317

.331

.338

.354

.363

.372

Increase

.029

.061

.075

.082

.098

.107

.116

Bottle No. 11:
Acidity

.308

.356

.368

.395

.394

.423

.432

.451

Increase

.048

.06

.087

.086

.115

.124

.143

Bottle No. 12:
Acidity

.349

.427

.423

.46

.428

.492

.54

.543

Increase

.078

.074

.111

.079

.143

.191

.194

Bottle No. 13:
Acidity

.41

.46

.45

.46

.497

.515

.561

.672

Increase

.005

.04

.05

.087

.105

.151

.262

Bottle No. 14:
Acidity

.456

494

.519

.552

.528

.599

.68

.708

Increase

.038

.063

.096

.072

.143

.224

.252

From the conditions of the experiment it is to be expected that
the increase of acidity in the raw milk would be greater than in the
heated milk, and that if the bacterial enzyms were operative the
increase in acid would be directly proportional to the amount of
enzym present or to the mass of cells. It will be noted that these
are the conditions shown to be present by the figures given in the
table. The data from three bottles of each of the raw and heated
milks are given in graphical form in figures 1 and 2. The bottles

INCREASE OF ACIDITY IN MILK.

CENT ' -OLUOL

a i3a K 4 130

FIG. 1. Increase of acidity in raw milk preserved with 3 per cent toluol. Bottle 1 con-
tained the minimum number of bacteria, bottle 7 the maximum, and bottle 4 an
intermediate number.

CENT
AClQlT-l

HEA

OLUOL

4 90 So 105 I IS HA

126 113 133

FIG. 2. The increase of acidity in heated milk preserved with 3 per cent toluol. Bottle 8
contained the minimum number of bacteria, bottle 14 the maximum, and bottle 11 an
intermediate number.

BACTERIOLOGY OF CHEDDAR CHEESE.

selected were the ones containing the minimum and maximum
numbers of bacteria and an intermediate bottle from each set. The
curves presented show the results to be entirely consistent with what
might be expected.

In order to make the comparisons more easy there are given in
Table 14 the rate of daily increase for each bottle of the two sets.
The difference between the increase of acid in the raw milk and the
heated milk should give the increase in the raw milk due to the
inherent enzyms of the milk. This increase should be constant, or
nearly so, since the same amount of enzym was operative in each
bottle, although under somewhat different conditions, as regards
acidity in the different bottles.

TABLE 14. Daily increase in acidity in bottles of raw and heated milk preserved

with toluol.

Raw milk.

Heated milk.

Increase
(due to
enzyms
of milk).

Bottle 1

Per cent.
0.0012

Bottle 8

Per cent.
0005

Per cent.
0007

Bottle 2

.0014

Bottle 9

.0005

0009

Bottle 3

.0018

Bottle 10. . .

.0007

0011

Bottle 4

.0015

Bottle 11

.0010

0005

BottleS

.0020

Bottle 12

0013

0000

Bottle 6

.0023

Bottle 13

.0017

0007

Bottle?

.0027

Bottle 14.

.0017

.0016

It will be noted that the daily increase is proportional to the
amount of bacteria present, and that the daily increase due to the
inherent enzyms of milk is as constant as could be expected. The
comparison has been made bottle for bottle in the two series. This
introduces an error, since the amount of bacterial cells present in
the compared bottles of raw and heated milk was not always the
same. In the case of bottles 2 and 9, which are compared, the initial
acidity was identical, while in the case of bottles 6 and 13^ which are
also compared, the initial difference in acidity was 0.083 per cent.
The results seem to leave no doubt concerning the presence of an acid-
forming enzym in the organisms of the Bacterium, lactis acidi group
that acts on the milk sugar. It might be thought that the increase
in acidity was due to the production of amino acids by a proteolytic
enzym. In this case the soluble nitrogen must be augmented. That
k this does not occur has been shown by numerous investigators.

The experiment was repeated in full detail with identical results.
The increase in acid can not be asserted to be due to the formation of
lactic acid, since, of course, no qualitative tests could be made in the
presence of the lactic acid present in the milk at the beginning of
the experiment.

OTHER GROUPS OF BACTERIA IN CHEDDAR CHEESE. 31

It is true that in cheese the sugar disappears within a few days,
and the enzym which was active under the conditions of the experi-
ment could therefore not be' active in cheese. This group of organ-
isms has been believed to be peculiarly deficient in enzyms. It has
even been claimed that they were the only bacteria devoid of cata-
lase; no proteolytic enzyms have been demonstrated in them, and
heretofore none acting on carbohydrates with the formation of acid.
It is very probable that various classes of enzyms are formed by the
lactic bacteria. As has been shown, a considerable part of the mass
of the cheese consists of the cells of these organisms, which slowly
disintegrate and their intracellular products are set free. It is not
at all improbable that these products are the casual agents of changes
that occur in the cheese, and that the roles of the lactic bacteria are
not limited to those previously mentioned.

OTHER GROUPS OF BACTERIA IN CHEDDAR CHEESE.

It is difficult to conceive that the varied chemical changes that
occur during the ripening of Cheddar cheese can be due directly or
indirectly to the lactic bacteria alone. It seems as though other
biological factors must be operative, but, as was previously stated,
no one has demonstrated the constant occurrence in large numbers
of any other group of bacteria than the lactic group in Cheddar
cheese. With the purpose of making a more complete examination
of the cheese during the entire ripening period, the plate-culture
work has been supplemented by other methods.

DETERMINATIONS BY MILK-DILUTION AND PLATE-CULTURE METHODS.

Since no solid medium seemed to promise better results than the
standard media hitherto employed, milk was chosen as the medium
most likely to permit of the growth of other groups of bacteria
possibly present. In a previous publication 1 data concerning the
distribution of a group of rod-shaped lactic bacteria in milk and
other dairy products have been given. Among these organisms are
included those found in many fermented milks, the Bacillus bul-
garicus group, also the B. casei group, to which the work of Von
Freudenreich and Jensen has attracted attention, as well as many
of the acidophilous organisms found especially in the alimentary
tracts of animals. These organisms were found to be constantly
present in milk, butter, and in all the samples of Cheddar cheese
examined. No quantitative analyses of cheese were made, however,
in the work to which reference has been made.

1 Hastings, E. G. ; Hammer, B. W. ; and Hoffman, C. Studies on the bacterial and
leucocyte content of milk. Wisconsin Agricultural Experiment Station, Research Bulletin
6. Madison. June, 1909.

32 BACTEEIOLOGY OF CHEDDAE CHEESE.

Von Freudenreich's work showed the constant presence in large
numbers of the lactic bacilli in Emmental cheese. In the making
of this cheese they are added to the milk in great numbers in the
natural whey rennet employed^ and the high temperature of the
curd during the pressing of the cheese favors their development.
These organisms find a more favorable condition for growth in milk
than in any of the usual media, indeed some of the members of the
group can not be cultivated except in milk.

With the idea of determining the number of the organisms of
this group in cheese, inoculations in varying dilutions were made
from cheese emulsions into flasks of sterile milk, which were pro-
tected from evaporation by tin-foil caps and incubated at 37 C.
The dilutions increased by a ratio of 10, thus flasks of milk in the
case of a single cheese might be inoculated with amounts of a cheese
emulsion representing 0.001, 0.0001, and 0.00001 gram. The extent
to which the dilutions were carried depended on the age of the
cheese. An attempt was constantly made to carry the dilutions to a
point where some of the flasks would remain sterile. The method
is thus a quantitative one for bacteria that will grow under the
conditions obtaining. The dilution method is to be considered a
rough way of determining the number of certain classes of bacteria.

After incubation for one month at 37 C., the acidity of each flask
was determined. The Bacterium lactis acidi group of organisms
produce in average milk an acidity ranging from 0.7 to 1.25 per cent.
The lactic bacilli produce an acidity usually exceeding 1.25 per cent.
It has been the practice to infer that every flask of milk showing an
acidity above 1.25 per cent after one month's incubation contains the
lactic bacilli, either in pure culture or mixed with organisms of the
Bacterium lactis acidi group. In the flasks showing an acidity less
than 1.25 per cent it can not be inferred that the lactic bacilli are
absent, since some cultures are found that produce no more acid than
Bacterium lactis acidi. Many microscopical examinations were made
of the flasks to establish the accuracy of the conclusions drawn from
the data obtained by titrations. Microscopic examinations were also
made of all the flasks from which the titrations did not give con-
clusive evidence concerning the organisms present. The smears were
stained with Gram's stain and decolorized with a mixture of one part
of anilin oil and two parts of xylol. This method of decolorizing
removes the stain from the casein, but not from any of the organisms
that have been found in cheese.

The dilution method thus gives information concerning the ab-
solute and relative number of acid-forming bacteria of these two
groups. It may also give information concerning the presence of
still other types of bacteria when they are present in greater numbers
than those of the groups already referred to, since they will then
appear in the flasks in pure culture.

NUMBEE OF BACTERIA IN CHEESE.

In Table 15 are given the results of the analysis of four cheeses.
A portion of the data has been presented in Table 2 and is here
repeated for ease of comparing the numbers of bacteria as determined
by plate-culture methods and by the dilution method. It will be
noted that the dilution method, as a rule, gives higher results than
the plate culture. Out of 49 determinations, the dilution method
gave higher results than the lactose-agar plate determinations in 30
cases. It is probable that the dilution method always gives higher
results. If the results show a growth in a dilution of 1 to 10
millions, while the dilution of 1 to 100 millions gives a negative
result, it can only be asserted that the bacterial content of the
cheese was between 10 and 100 millions. It will be noted that
the dilution method often shows many fold more bacteria than
the plate cultures, and that the reverse is net often true. In
cases where the latter gives the higher number, the excess is not
great, only three or four times greater. The only' explanation that
can foe given of the higher numbers obtained by the dilution method
is that some types of bacteria present in the cheese that do not appear
on the plate cultures find conditions favorable for growth in the milk.
The results obtained by the dilution method indicate that other
organisms than the Bacterium laptis acidi group are present in the
cheese, undoubtedly in great numbers.

TABLE 15. dumber of bacteria per gram of cheese as determined on lactose
agar and gelatin plates, and in flasks of milk inoculated with high dilutions
of cheese.

[Numbers expressed in millions.]

Age.

Cheese No. 1.

Cheese No. 54.

Cheese No. 91.

Cheese No. 92.

Lac-
tose
agar.

Gela-
. tin.

Milk
dilu-
tion.

Lac-
tose
agar.

Gela-
tin.

Milk
dilu-
tion.

Lactose
agar.

Gelatin.

Milk
dilution.

Lac-
tose
agar.

Gela-
tin.

Milk
dilution.

Days.
2
4
8
11
14
22
30
37
45
56
77
86
100
113
124
143
155
165
176
187
208

150

1,000
10,000
1,000
100
10,000
1,000

250

150

100
1,000
100
100
1,000
10,000

120

400

340

1,000

1,400
58

530
1,600

500
35

2.5
2

10
10

1,000
1,000

10
100

270
1,400
1,300

480

1,000
100
100

1,000
1,000
10

340
225

100
225

32
51

10
1,000

250

36.5

128
74
145
50
18
15
17.5

320
65
142
28.5
23.5
10.5
5.5

100
100
100
100
100
10
100

250
100
132
145
38.5
63
9

84
120
87
96
38
32
5.5

100
1,000
1,000
100
10
100
10

8.5

14
22.5

12
12.5

10
100

3.5

2.5

100

7.5

12.5

100

1.5

From the results obtained from the determinations of the degree
of acidity attained by the flasks of milk inoculated with varying

BACTERIOLOGY OF CHEDDAR CHEESE.

amounts of cheese emulsion, and from the microscopic examinations
of the same flasks, it has been possible to determine the relations
existing between the different groups of acid-forming organisms, as
well as their absolute numbers.

Table 16 has been constructed from such data. For ease in com-
parison the same data are expressed in Table 17 in percentages, and
two additional cheeses are also included in 1 the latter table. It will
be noted from the data presented in Table 17 that during the early
part of the ripening period the organisms of the Bacterium lactis
acidi group make up over 90 per cent, and in many cases approxi-
mately 100 per cent, of the acid-producing flora of the cheese. With
increasing age the ratio changes, until late in the ripening period
the rod-shaped lactic bacilli predominate, and in many cases make
up over 90 per cent of the acid-forming bacteria found in the cheese.

It will also be noted from the data given in Table 15 that the period
at which the maximum number of bacteria is found, as determined
by the dilution method in milk, is coincident with the appearance of
the lactic bacilli, as shown in Table 16. Using any culture medium
that furnishes favorable conditions for the growth of both groups
of lactic bacteria, the maximum number of organisms should be
found at the time when the group first to appear the Bacterium
lactis acidi group has attained its maximum development, but be-
fore any considerable number of the cells have died, and at the time
when the second group of organisms finds favorable conditions for
growth in the cheese.

TABLE 16. Numbers of Bacterium lactis acidi and of acid-producing bacilli in
cheese as determined in milk cultures.

[Numbers expressed in millions.]

Age.

Cheese No. 1.

Cheese No. 54.

Cheese No. 85.

Cheese No. 91.

Cheese No. 96.

Bact.
lactis
acidi.

Acid-
pro-
ducing
bacilli.

Bact.
lactis
acidi.

Acid-
pro-
ducing
bacilli.

Bact.
lactis
acidi.

Acid-
pro-
ducing
bacilli.

Bact. lac-
tis acidi.

Acid-
pro-
ducing
bacilli.

Bact. lac-
tis acidi.

Acid-
pro-
ducing
bacilli.

Days.
2

1,000

10,0<)0
1,000
100
10,000
1,000

10
100

100
1,000
100
10,000
1,000

11....
14.. .

1,000

""io"

100
10
1,000
100

10
10

""io"

100
10

10
10
10
10
10
1,000

1,000

""io"

1
100

1
1

22....
30....
37

1,000
.100

1,000
100

1,000
100
100

100
100
100

100
1,000
100
100
100
100
100
100
100
10

10
10
10

1
1

10
10

45....
66

100

86....
100...

100
100
100
100
100
10
100

100
100
10
10
10
10
100

10
10
10

113...

124. .

10
100

143. . .
155..

165

176...

187.

100
10

100

208

RESULTS OF PLATE CULTURES.

TABLE 17. Relative proportion of the Bacterium lactis acidi type and the lactic
acid-producing bacilli, as determined l>y dilution cultures in milk.

Age.

Cheese No.
1.

Cheese No.
64.

Cheese No.

58.

Cheese No.
85.

Cheese No.
91.

Cheese No.
92.

Cheese No.
96.

Bact.
lactis
ac*di.

Acid-
pro-
duo-
ing
ba-
cilli.

Bact.
lactis
acidi.

Acid-
pro-
duc-
ing
ba-
cilli.

Bact.
lactis
acidi.

Acid-
pro-
duc-
ing
ba-
cilli.

Bact.
lactis
acidi.

Acid-
pro-
duc-
ing
ba-
cilli.

Bact.
lactis
acidi.

Acid-
pro-
duc-
ing
ba-
cilli.

Bact.
lactis
acidi.

Add-
pro-
duc-
ing
ba-
cilli.

Bact.
lactis
acidi.

Acid-
pro-
duc-
ing
ba-
cilli.

Days.
4
8
11
14
22
30
37
45
66
77
86
100
113
124
143
155
165
176
187
208

P.ct.

90.9

P.ct.
9.1

P.ct.

99.9
90.9

P.ct.
0.1

9.1

P.ct.

99.9
99
99.9

P.ct.
0.1
1
.1

P.ct.

90.9

9.1

90.9

9.1

99.99
90.9

.01
9.1

99.9
99.9

.1
.1

99.99
99.9

0.01
.1

60
50

50
50

9.1
50
99
9.1
99
99
50
60
50
50
1

90.9
50
1
90.9
1
1
50
50
50
50
99

99
50

90.9
50

9.1
50

90.9
90.9
50
90.9
50
90.9
50
90.9
50
90.9
50

9.1
9.1
60
9.1
60
9.1
60
9.1
50
9.1
50

90.9
99.9
90.9
99
90.9
90.9
90.9
99
99
60
1

9.1
.1
9.1
1
9.1
9.1
9.1
1
1
50
99

90.9
99

9.1
1

9.1

90.9

50
50
90.9
90.9
90.9
60
50

50
60
9.1
9.1
9.1
50
50

90.9

9.1

90.9
90.9

9.1
9.1

90.9
90.9

1
1
50

99
99
50

60
.1

50
99.9

99
1

Since some of the lactic bacilli develop on lactose agar plates, it
was thought that if all of the colonies on a certain portion of each
plate were inoculated into milk one should obtain information con-
cerning the sequence of development of the different groups of lactic
bacteria in the cheese and also concerning the ratio existing between
them.

For this purpose an area showing well-isolated colonies has been
circumscribed, and every colony within the area has been inoculated
into milk. It has been inferred that all tubes that curdled within
48 hours at 37 C. contained Bacterium lactis acidi, while those that
curdled between the second and tenth day contained the lactic bacilli.
Enough control work was done to show that this method of differen-
tiation is sufficiently accurate for the purpose in hand. The tubes
that did not curdle in 10 days were examined microscopically to
determine the organism present.

Six cheeses have been thus examined at frequent intervals during
the period of ripening. The data are given in Table 18. It will be
noted that the results are confirmatory of those obtained by the dilu-
tion method in milk, although not so striking, since many of the
lactic bacilli do not develop on the plate cultures. The period at
which the lactic bacilli appear is later than in the previous exami-
nations (Table 16), which demonstrated their presence in consider-
able numbers within the first week of the ripening period. The pre-
vious examinations were made with dilution cultures in milk. In

BACTERIOLOGY OF CHEDDAR CHEESE.

such the lactic bacilli may make themselves evident when they are
present in very small numbers as compared with the Bacterium lactis
acidi. With the plate-culture method, unless they are present in
considerable numbers, their presence in the cheese is not likely to be
detected. This error in the method has been previously pointed out.
In the plate cultures they are likely to be missed unless they are pres-
ent in a ratio of 1 to 10 of Bacterium lactis acidi.

The same cheeses have also been examined by the dilution method
in milk. The results are given in Table 19. It will be seen that
they are confirmatory of the previous analyses.

Tlie great delicacy of the dilution method as a means of detecting
the lactic bacilli in the presence of Bacterium lactis acidi is shown
in the results given in Table 19. On the examination of cheese 308C
at two days the percentage of Bacterium lactis acidi is given as
99.9999 and that of lactic b.acilli as 0.0001. By this is meant that
Bacterium lactis acidi was detected in dilutions approximately one
million times greater than the lactic bacilli. For example, the acidity
of the flask inoculated with one ten-thousandth gram of cheese was
1.64 per cent, thus indicating the presence of lactic bacilli, while the
flask inoculated with one ten-billionth gram of the cheese had an

acidity of 0.87 per cent, indicating Bacterium lactis acidi.

TABLE 18. The relative proportions of Bacterium lactis acidi and lactic bacilli
in cheese as determined ~by plating on lactose agar.

Age.

Cheese No.
307C.

Cheese No.
308C.

Cheese No.
309C.

Cheese No.
310C.

Cheese No.
311C.

Cheese No.
312C.

Bact.
lactis
acidi.

Lactic
bacilli.

Bact.

lactis
acif.

Lactic
bacilli.

Bact.
lactis
acidi.

Lactic
bacilli.

Bact.
lactis
acidi.

Lactic
bacilli.

Bact.
lactis
acidi.

Lactic
bacilli.

Bact.
lactis
acidi.

Lactic
bacilli.

Milk before ren-
net was adaed
When put in
ipress

P.ct.
100

100

100
100
100
100

P.ct.

P.ct.
100

100

100
100
100
86

P.ct.

P.ct.
100

100

100
100

P.ct.

P.ct.
100

100
100

P.ct.

100

100
100
100
100

P.ct.

100

100
100

P.ct.

When taken
from press
2 days

3 days

4 days

90
100

100
100

6 days

100
100
86
90
100
80
100

14
10

7 days ... ....

100
100
100
100
100
100

100
100
100
90
100
100
70

100
100

9 days

11 days ...

100
86

14 days

100
33

100
90

18 days .

21 days... .

22 days

100

28 days ..

100

33 days

36 days ..

100
100
100
00
90

10
10

42 days

49 days

29
40

71
60

56 days

64 days

RESULTS OP PLATE CULTUEES.

TABLE 19. The ratio between the numbers of Bacterium lactis acidl and the
lactic bacilli at different stages in the ripening of Cheddar cheese, as deter-
mined by milk-dilution method.

Age.

Cheese No.
307C.

Cheese No.

308C.

Cheese No.
309C.

Cheese No.
310C.

Cheese No.
311C.

Cheese No.
312C.

Bad.

lactis
acidi.

Acid-
pro-
duc-
ing
ba-
cilli.

Bact.
lactis
acidi.

Acid-
produc-
ing
bacilli.

Bact.
lactis
acidi.

Acid-
ro-
ue-
ing
ba-
cilli.

Bact.
lactis
acidi.

Acid-
pro-
duc-
ing
ba-
cilli.

Bact.
lactis
acidi.

Acid-
pro-
due-
ing
ba-
cilli.

Bact.
lactis
acidi.

Acid-
pro-
duc-
ing
ba-
cilli.

Milk before
rennet was
added

Perct.

Perct.
99.99

Perct.
0.01

Perct.

Perct.
99

99.99

Perct.
1

.01

Perct.
99

Perct.

Just before
put in press
When taken
from press. .
2 days

99.999

99.99
99.9999
99
99.9

"go.'g""

90.9
90.9
50
90.9
50
99
90.9

.001

.01
.0001
1
.1

"9.Y"
9.1
9.1
50
9.1
50
1
9.1

99.9

99.9
99

.1
1

99.99
99.99
99.9
99.99
99.99

0.01
.01
.1
.01
.01

99
99

99.9
50
99
99.99

0.1
50
1
.01

99
90.9
99.99

1
9.1
.01

3 days

4 days ....

99.9
99
99.9
99
50
50
90.9
50
90.9
50
50

.1
1

1
50
50
9.1
50
9.1
50
50

5 days
6 days ...

99.9
90.9

.1
9.1

7days

90.9
50
99
99
50
90.9
50

9.1
50
1
1
50
9.1
50

99.9
99

.1
1

90.9
99

9.1
1

11 days ....

90.9

9.1

14 days

90.9

9.1

18 days

21 days

28 days ..

33 days

35 davs.

50
50

50
99

42 days

90.9

9.1

It must not be inferred that the figures given in the tables repre-
senting the results obtained by the dilution method indicate the ex-
act number of the different groups of lactic organisms or the exact
ratio existing between them. The sudden increase or decrease of the
ratio is due to the inherent errors of the dilution method. It would
be an endless task to show by this method, or any other, in fact, the
exact proportion between the different types of organisms in the
cheese at various stages in the ripening period.

From the data presented there would seem to remain no doubt that
the lactic bacilli develop later than the Bacterium lactis acidi group,
making their appearance within a week or 10 days after the cheese
is made, gradually increasing in numbers and probably attaining
their maximum during the first month and then gradually decreasing
in numbers. The number found per gram of the cheese ranges from
a few million to one billion. Usually they do not attain such great
numbers as do the ordinary lactic bacteria. Again, their numbers
may equal the lactic bacteria, as in cheese No. 1, Table 16.

DETERMINATIONS BY MICROSCOPIC EXAMINATION OF THE CHEESE.

It was also thought that the gradually changing flora should mani-
fest itself in the appearance of microscopic preparations made from
the cheese. At the various periods of sampling smear preparations

BACTEEIOLOGY OF CHEDDAR CHEESE.

were made from the emulsions and stained with Gram-Weigert's
stain as described on page 32.

The preparations show in a general way the same change in flora
as has already been made evident by the analyses presented. It is
not to be expected that the microscopic examination would give re-
sults as striking as the cultural, since the latter measures the living
cells, the former only those cells that have not lost their staining
properties.

2, DAYS

57 DAYS

I \Z DAYS 1 89 DAYS

PIG. 3. Typical microscopic fields from cheese No. 54 at different stages in the ripening
process. \ Camera lucida drawings.)

Figures 3 and 4 have been prepared from camera-lucida drawings
of typical microscopical fields of several slides made from 2 cheeses.
The results of the cultural examination have been presented in
Tables 15 and 16. Figure 3 shows that at 2 and 57 days the organ-
isms are almost, if not wholly, of B.acterium lactis acidi group; at
112 days there is a preponderance of Bacterium lactis acidi and a few
lactic bacilli, while at 18? days but few Bacterium lactis acidi cells

RESULTS OP MICROSCOPIC EXAMINATIONS.

remain, the lactic bacilli having become more evident. Figure 4 in-
dicates likewise that at 29 and 78 days the organisms are wholly of
the Bacterium lactis acidi group, while at 121 and 185 days a decrease
is seen in Bacterium lactis acidi and an increase, in lactic bacilli. It
will be noted that rod-shaped organisms do not appear nearly as
soon as would be indicated by the cultural analyses, but that they
do at last appear, especially when the cells of the lactic bacteria have
greatly decreased in numbers. This may again be taken as evidence

29 DAYS

7G DAYS

131 DAYS IS5DAY5

PIG. 4. Typical microscopic fields from cheese No. 91 at different periods in the ripening
process. (Camera lucida drawings.)

that the lactic bacilli develop subsequent to the Bacterium lactis
acidi group and that they tend to disappear less rapidly.

DISAPPEARANCE OF BACTERIAL CELLS IN CHEESE.

The drawings presented in figures 3 and 4 are evidence of the grad-
ual disappearance and disintegration of the bacterial cells in cheese.
It is a well-known fact that the enzyms elaborated by bacteria in
pure cultures continue to act long after the death of the cells. There

40 BACTERIOLOGY OP CHEDDAR CHEESE.

are many reasons for believing that there is no enzym action until
cell disintegration begins. It has been previously shown that the
Bacterium lactis acidi group of bacteria elaborate enzyms that are
able to increase the acidity of milk. As previously indicated, it may
be surmised that still other types of enzyms are formed by this group
of bacteria. The direct influence of the immense number of acid-
forming bacteria, amounting to billions per gram, must be of great
importance in cheese ripening; the indirect action through their
enzyms may be still greater.

It is certainly true that in normal Cheddar cheese the period of
development of the ordinary lactic bacteria the organisms which
heretofore have been believed to be the only ones of importance in
cheese because of their constant presence in great numbers is fol-
lowed by the development of another group of organisms, which,
while they ferment milk sugar, producing lactic acid, must have
some other source of carbon in cheese on account of the total disap-
pearance of the sugar before their maximum period of development.
The influence of the first group has been pointed out early in this
paper. The role of the second group can not at this time be de-
termined, but because of their constant presence in numbers, approxi-
mating if not equaling those of the first group, their influence on
the ripening of the cheese can not be a minor one.

DETAILED STUDY OF LACTIC BACILLI.

The work of Von Freudenreich and numerous later investigators
indicates that the lactic bacilli represent a group with certain com-
mon characteristics, such as morphology, optimum temperature for
growth, and to some extent in the production of compounds from the
sugar fermented. In all fields of bacteriology it is difficult at the
present time to draw the boundary lines of any group of bacteria.
The placing of the lactic bacilli found in cheese in a single group
may be incorrect, but because of the fact that all the cultures studied
have certain characters in common, it seems best to discuss them as
a single group, although more detailed study might divide them into
a number of groups. A comparative study has been made of 20 pure
cultures isolated in the course of the work! The main facts of this
study are here presented. All the cultures were isolated from cheese
with the exception of No. 160, which was obtained from milk. Cul-
tures 58A and 58B represent different colonies from plates made to
insure the purity of the cultures to be used in the detailed study.
The differences noted in the study of these two cultures originally
from the same culture illustrate the differences one may expect to
obtain in cultural work.

The action of the different cultures in milk is given in Table 20, in
which they have been arranged according to the degree of acidity
produced in milk.

DETAILED STUDY OF LACTIC BACILLI. 41

ACIDITY PRODUCED.

It will be noted from the data in Table 20 that both the rate of
acid development, and hence the time required to curdle milk, as
well as the maximum amount of acid produced, varies widely, the
acidity varying from 0.91 per cent to 2.31 per cent. The acidity
produced by each culture increased after the tenth day. Since the
flasks were protected by tin-foil caps, this increase can not have been
due to evaporation.

White and Avery, 1 in studying cultures from fermented milks,
noted the same differences in acid production. They established
two groups, one producing an acidity in milk of about 1 per cent,
the other of 3 per cent, in 10 days. Such a division of the cultures
studied could not be made.

Rogers 2 states that a typical culture of the lactic bacilli from
fermented milk produces nearly 3 per cent of acid in three days at
37 C. Von Freudenreich and Thoni 3 studied cultures from Em-
mental cheese that produced from 0.2 to 1.26 per cent of acid in
whey to which peptone had been added.

Out of the several hundred titrations made of milk inoculated
with cheese emulsions but 15 showed an acidity above 2 per cent.
When raw milk is incubated at 37 C., the acidity will often, if not
in the majority of cases, exceed 2 per cent and at times may reach
3.5 to 4 per cent. It would thus seem that certain lactic bacilli pres-
ent in milk do not find favorable conditions for development in
Cheddar cheese.

FORMS OF LACTIC ACID PRODUCED.

A number of investigators have determined the rotary power of
the lactic acid formed by the lactic, bacilli. Bertrand and Weis-
weiller* concluded that the acid was a mixture of the levo and
dextro forms, with a slight predominance of the latter.

1 White, Benjamin, and Avery, Oswald T. Observations on certain lactic acid bacteria
of the so-called Bulgaricus type. Centralblatt filr Bakteriologle, Parasltenkunde und
Infektionskrankheiten, Abteilung 2, vol. 25, No. 5/9, pp. 161-178. Jena, Nov. 30, 1909.

2 Rogers, L. A. Fermented milks. United States Department of Agriculture, Bureau
of Animal Industry, Twenty-sixth Annual Report (1909), pp. 133-161. Washington, 1911.
Reprinted as Bureau of Animal Industry Circular 171, Washington, 1911.

3 Von Freudenreich, Edward, and ThSni, J. Sur 1'action de dlffe'rents ferments lac-
tiques sur la maturation du fromage. Revue Ge'ngrale du Lait, vol 4, No. 8, pp. 169-181,
Jan. 30; No. 9, pp. 200-209, Feb. 15; No. 10, pp. 225-232, Feb. 28; No. 11, pp. 247-259,
Mar. 15. Lierre, 1905.

* Bertrand, Gabriel, and Weisweiller, Gustav. Action du ferment bulgare sur le lalt.
Annales de Institut Pasteur, vol. 20, No. 12, pp. 977-990. Paris, Dec. 25, 1906.

BACTEBIOLOGY OF CHEDDAE CHEESE.
TABLE 20. Action of lactic bacilli in milk.

Cul-
ture
No.

Acidity pro-
duced in milk.

Rotation of acid.

Time
of cur-
dling.

Cul-
ture
No.

Acidity pro-
duced in milk.

Rotation of acid.

Time
of cur-
dling.

10 days.

45 days.

10 days.

45 days.

5
152
52
165.1
163
58A
58B
128
116
120
104

Pact.

0.324
.643
.68
.69

Per ct.
0.91
1.12
1.05
1.10
1.26

Days.
11

116.2
92
160
165
113
71
91
85
116.1
96

Per ct.
1.2
1.24
1.37
1.41
1.57
1.6
1.68
1.73
1.75
1.89

Per ct.
1.44
1.4
1.6
1.57
1.88
1.75

Inactive...

Day*.

Inactive

4
4

4
4
4
4

Dextro

.89
.86
.89
.81
1.02
1.17

1.38
1.21
1.2
1.36

Inactive+levo

1.89
1.96
2.31

Dextro+ inactive

Inactive

1.46

Dextro+ inactive

Heinemann and Hefferan 1 report the formation of an inactive
acid, while White and Avery 2 found that the organisms that pro-
duced a large amount of acid, 3 per cent, formed an inactive acid,
whereas the cultures that formed acid slowly and in smaller amounts
showed a levo-rotary power at the end of 24 hours, but in older
cultures there were approximately equal amounts of levo and of
inactive acids.

The rotary power of 9 of the cultures studied was determined by
Dr. J. M. Currie. Four of these produced inactive acid ; 1 produced
inactive and levo acid, with a predominance of the inactive form;
2 produced a mixture of dextro and inactive acids, with a predomi-
nance of the dextro-rotatory form; and the remaining 2 produced
pure dextro acid. One culture isolated from milk, of which no other
study was made, produced pure levo acid. With 11 cultures isolated
from other sources than milk or cheese, only the pure dextro or pure
inactive acids were found.

This type of organism, then, can produce inactive acid, or active
acid of either modification, or a mixture of the inactive acid with
either of the active acids. All of these forms were found in cultures
isolated from Cheddar cheese, with the exception of the pure levo
acid.

TYPE OF CURD PRODUCED.

In most of the more active cultures the reduction of litmus and the
return of color proceeded in exactly the same manner as in a litmus-
milk culture of Bacterium lactis acidi. On the other hand, curdling
took place with only a slight or partial reduction of the litmus in
some cultures.

1 Heinemann, P. G., and Hefferan, Mary. A study of Bacillus Bulgarlcus. Journal of
Infectious Diseases, vol 0, No. 3, pp. 304-318. Chicago, June 12, 1909.

2 White, Benjamin, and Avery, Oswald T. Observations on certain lactic acid bacteria
of the so-called Bulgarlcus type. Centralblatt filr Bakterlologle, Parasltenkunde und
Infektlonskrankhelten, Abtellung 2, vol. 25, No. 5/9, pp. 161-178. Jena, Nov. 30, 1909.

DETAILED STUDY OF LACTIC BACILLI.

There was a slight gas formation in most of the cultures, evidence
of which was found in the slight furrows or tiny holes which some-
times appeared in the curd. These gas holes were, however, not
always found in curds from the same culture. Although most of the
organisms grew in lactose-agar shake cultures, no gas formation was
noted in any case.

FERMENTATION OF SUGARS.

Sixteen of the cultures were tested as to their power to ferment
sugars. The cultures were incubated eight days at 37 C. The test
for growth and fermentation of the sugar was the reddening of
litmus paper. The results are given in Table 21. All of the strains
tested except one, No. 128, grew in glucose bouillon. Many of the
strains made no growth or only a feeble growth (indicated by the
sign ) in maltose and sucrose bouillon, although all except No. 128
grew in one or the other, or both, of these sugars.

TABLE 21. Growth of lactic bacilli in culture media.

Cul-
ture
No.

Growth in sugar
bouillon.

Growth
on lac-
tose
agar.

Growth
on or-
dinary
gelatin.

Cul-
ture
No.

Growth in sugar
bouillon.

Growth
on lac-
tose
agar.

Growth
on or-
dinary
gelatin.

Glu-
cose.

Mal-
tose.

Su-
crose.

Glu-
cose.

Mal-
tose.

Su-
crose.

5
152
52
165.1
163
58A
58B
fl28
116
120
104

+ + + -H + + + 1 + + -H

+
+

116.2
92 .
160
165
113
71
91
85
116.1
96

+
+
+
+
+
+
+
+
+
+

+
+
+

+
+

+
+
+
+

+
+

++++++ 1

+ + + H- + + H-

+
+

+
+
+

+
+

indicates feeble growth.
FORM OF COLONIES.

The growth upon lactose agar and ordinary gelatin was deter-
mined in plate cultures on the same media that was used for the
bacteriological analysis of cheese. Growth was obtained in lactose-
agar plate cultures from every strain except No. 128. There was a
good growth in most of the cultures, although with some only a few
colonies developed. When the plates were incubated for several
days the colonies continued to increase in size, sometimes attaining
a diameter of 1 mm. The surface colonies are round and the deep
colonies have the common flattened elliptical form. They can not
be distinguished from the colonies of Bacterium lactis acidi. This is
probably one reason why previous investigators have failed to find
this type of organism in Cheddar cheese.

Growth upon plates of ordinary gelatin was obtained from 8 of
the cultures, This, however, was not constant for some strains

44 BACTEKIOLOGY OF CHEDDAR CHEESE.

which developed from one inoculation failed to grow from another
inoculation. The variable growth of this type of organism in plate
cultures can account for some of the inconsistent results obtained in
the quantitative analysis of cheese by means of lactose-agar and gela-
tin plates.

The readiness with which some of these cultures of lactic bacilli
isolated from cheese grow upon ordinary media is rather surprising,
in view of what has been stated by many authors concerning this
property. One of the characteristics generally given for this group
of organisms is their meager growth upon ordinary media. The five
cultures studied by Von Freudenreich failed to grow on ordinary
gelatin or on gelatin to which an extract of cheese had been added.
Cohendy * and Lohnis 2 found these organisms hard to cultivate be-
cause of their reluctance to grow upon the ordinary nutrient media.
White and Avery state that when freshly isolated from their natural
environment milk they do not develop on any of the usual nutrient
media, even though sugar be present. Heineman and Hefteran, on
the other hand, state that they grow well in milk, in media prepared
from milk, or if glucose is added to the ordinary media. All of the
cultures studied were isolated by means of lactose-agar plates with
the exception of No. 128, which was obtained in pure culture in the
dilution cultures in milk.

THERMAL DEATH POINT.

The resistance of the cultures to heat was determined on 3-day -old
milk cultures which had been diluted and shaken with water and
then drawn into sterile capillary tubes. The tubes were held for 30
seconds in water at various temperatures. Most of the cultures were
killed at some temperature between 62 and 67 C. Two cultures
were killed between 60 and 62.5 C., and two cultures were killed
at about TO C.

MORPHOLOGY.

The bacilli are nonmotile and do not bear spores. In a single mi-
croscopic field of a slide prepared from a pure culture the organisms
may vary in length from 2.5 microns to 20 or 30 microns or more.
Sometimes the filaments are very long. In one of the pure cultures a
filament was found 75 microns in length. In one of the mixed cul-
tures inoculated from cheese there was found a filament which was
about 9GO microns in length, extending four times across the field of

1 Cohendy, Michel. Essals d'accHmation mlcroblenne perslstante dans la cavlte" Intes-
tinale. Comptes Rendus Hebdomadaires des Stances de la Socl^te" de Blologle, vol. 60
(annee 58, tome 1), No. 7, pp. 364-366. Paris, Feb. 23, 1906.

L<5hnl8, F. Versuch elner Grupplerung der Milchsaurebakterien. Centralblatt ftir
Bakterlologle, Parasltenkunde und Infektlonskrankheiten, Abteilung 2, vol. 18, No. 4/6,
pp. 97-149. Jena, Mar. 14, 1907.

DETAILED STUDY OF LACTIC BACILLI. 45

the microscope. The filaments are apparently made up of a number
of individual cells which for some reason do not show the cell divi-
sions, for frequently chains of rods are found instead of filaments.
Occasionally in a mixed, culture from a cheese inoculation filaments
were found very much curled, or curled at one end, but attempts to
isolate a culture which would regularly produce curled filaments
always resulted in securing a culture with the usual morphology. No
curled filaments were ever observed in pure cultures. The width of
the cells in a pure culture seems to be fairly constant; in some cultures
the individual cells are all slender, in other cultures they are all com-
paratively thick. In different cultures the width varies from 0.4 to
1.3 microns. No branching forms have ever been observed.

When stained with methylene blue the ends of the cells may take
the stain much more deeply than the remainder of the cell, or the
deeply stained spots may be scattered irregularly in the filament.
Occasionally the deeply stained spots appear as tiny nodules on the
rod. These characteristic staining properties are less frequently ex-
hibited when stained by the Gram-Weigert method. In a weakened
culture filaments are sometimes found with some of the cells Gram
positive, some of them Gram negative, and other cells taking the stain
partially.

Although there is such a wide difference between the cultures in
regard to the production of acidity in milk, the other characteristics
do not differentiate a culture producing a low acidity from one which
produces a high acidity. The morphology, however, seems to bear
some relation to the acidity produced, those producing a high acidity
being more slender than those producing a low acidity.

CONDITIONS FOR GROWTH IN CHEESE.

It has been pointed out that in their development in cheese the
lactic bacilli follow the Bacterium lactis acidi group, and that the
greater part of the growth must occur after the disappearance of the
sugar. Since lactic acid is the principal by-product of the fermenta-
tion of the sugar, it might seem probable that the lactic bacilli would
make use of this as a source of carbon and of energy. Analyses of
cheese show no decrease in lactic acid 1 at the period of development
of the lactic bacilli. This indicates that this group of bacteria does
not act on the lactic acid.

The action of the pepsin of the rennet extract activated by the
lactic acid results in the formation of peptones from the paracasein.
The favorable effect of peptone on the lactic bacilli is shown in the

1 Suzuki, S. K. ; Hastings, E. G. ; and Hart, E B. The production of volatile fatty
acids and esters In Cheddar cheese and their relation to the development of flavor. Wis-
consin Agricultural Experiment Station, Research Bulletin 11. Madison, June, 1910.
See p. 135.

BACTERIOLOGY OP CHEDDAK CHEESE.

following experiment. Small flasks containing 100 c. c. of sterile
milk were inoculated with cultures Nos. 5, 92, 91, and 96. To one
flask of each set there had been added before sterilization 0.5 gram
of peptone, and to another flask 1 gram. The control flask received
no peptone. In Table 22 the acidity at 10 days and 45 days is given.
The results show clearly that milk to which peptone has been added
is a better medium than plain milk for the growth of this group of
organisms. This was especially true in the case of culture No. 5,
which usually curdled milk in about 11 days, but which curdled the
milk to which peptone had been added in two days more rapidly
than the most active cultures curdle plain milk. The time of curdling
was shortened by the peptone 3 days in the case of culture No. 91,
and 1 day in cultures Nos. 92 and 96. In every case the final
acidity at 42 days was considerably greater than in plain milk, and
in every case but one (culture No. 91, at 42 days) there was a greater
development of acidity in the milk containing 1 gram than in the
milk containing 0.5 gram of peptone.

Incidentally, this experiment shows that the cessation of growth
in milk when a certain percentage of acidity is reached, which is a
fairly constant percentage for each particular culture, is not brought
about by the antiseptic action of the acid, but by a lack of suitable
nitrogenous food.

TABLE 22. Percentage of acidity produced by lactic bacilli in milk to which

peptone has been added.

Culture
No.

Acidity in 10 days.

Acidity in 42 days.

Plain
milk.

Milk with
0.5 gram
peptone.

Milk with
1 gram
peptone.

Plain
milk.

Milk with
0.5 gram
peptone.

Milk with
1 gram
peptone.

92
91

Per cent.
0.324
1.24
1.C8
1.58

Per cent.
1.19
1.4

1.9
1.87

Per cent.
1.32
1.58
2.1
1.96

Percent..
0.855
1.4

Per cent.
1.35
1.56
2.42
2.13

Per cent.
2.34
1.77
2.29
2.25

1.93

The action of the enzyms present in the cheese curd brings about
an increasing amount of soluble nitrogenous compounds so that after
one month of ripening about 18 per cent of the total nitrogen is in
the form of water-soluble compounds. 1 This action must render the
curd a medium favorable to the growth of the lactic bacilli, and
particularly to those strains similar to culture No. 5, which grow so
slowly in milk, or fail to grow therein, as frequently happens.

The dependence of this group of organisms upon enzyms from
other sources is shown by growing them together with other types

1 Van Slyke, L. L., and Hart, E. B. Conditions affecting chemical changes In cheese-
ripening. New York Agricultural Experiment Station, Bulletin 23G. Geneva, July, 1903.
See p. 160.

DETAILED STUDY OP LACTIC BACILLI.

of bacteria. Marshall and Farrand * have shown that when Bac-
terium lactis acidi grows in milk together with certain other organ-
isms there is an associative action which accelerates the production
of acid and the proliferation of cells. This quickening action was
found to occur in the case of 57 per cent of the cultures grown in
association with Bacterium lactis acidi. A similar action, but more,
pronounced, takes place when the lactic bacilli grow in milk together
with certain other types of organisms.

Suspensions in water were made of a 48-hour milk culture of the
lactic bacillus No. 104 and also of 48-hour glucose bouillon cultures
of two different strains of liquefying bacteria, which were isolated
from poor cheese. This particular type of organism has not been
found in good cheese. Small flasks of milk were inoculated with
various dilutions of these suspensions, as given in Table 23. Since
the results from the two experiments were similar, the figures for
only one of them are given.

TABLE 23. Associative action of lactic bacilli and a liquefying organism.

Flask
No.

Inoculation with liquefler.

Inoculation with lactic bacilli.

Acidity
in 2 days.

Acidity
in 3 days.

Acidity
in 7 days.

I
II
III
IV
V
VI
VII

1 1,000

No inoculation.

Per cent.
0.17
.18
.26
.40
.36
.42
.31

Per cent.
0.17
.59
.85
.96
.91
.99
.62

Per cent.
0.34
1.44
1.53
1.44
1.42

1 1,000

1:10,000,000 .. .

1 1000

1:100,000

1 1,000

1:1,000

1 100,000

1:1,000

1 10,000,000

1:1,OCO

No inoculation

1:1,000

1.36

It will be seen that in 2 days the production of acidity was accel-
erated in the cultures which received an inoculation of the liquefier.
This was more pronounced in 3 days, but less pronounced in 7 days,
when the flask which received no inoculation of the liquefier showed
only a small amount of acidity less than the flasks inoculated with
both cultures.

Other experiments were made with cultures of coccus forms iso-
lated from cheese. The liquefying action of these was not pro-
nounced, yet they exerted a stimulating effect on the lactic bacilli.
Reference to the coccus forms found in cheese will be made later.

SOLVENT EFFECT ON MILK PROTEINS.

Von Freudenreich was the first to demonstrate the fact that lactic
bacilli exert a digestive effect. The same action 2 was shown for cul-

1 Marshall, Charles E., and Farrand, Bell. Bacterial associations In the souring of
milk. Michigan Agricultural Experiment Station, Special Bulletin 42. East Lansing,
Mar., 1908.

2 Hastings, E. G., Hammer, B. W., and Hoffman, C. Studies on the bacterial and
leucocyte content of milk. Wisconsin Agricultural Experiment Station. Research Bulletin
6. Madison, June, 1909. See p. 202.

BACTERIOLOGY OP CHEDDAR CHEESE.

tures isolated from milk and has also been demonstrated by more
recent investigators.

The solvent effect of 8 of the cultures studied has been determined
by the anatyses of milk cultures after 3 months' incubation. .The
results are given in Table 24.

TABLE 24. The solvent effect of lactic iKicilli on milk proteins.

Culture.

Soluble N.
in 100 c. c.
of milk.

Increase in soluble N
inlOOc.c. of milk.

Sterile milk

Gram.
0.064
.072
.076
.080
.081
.090
.092
.096
.104

Or am.

o.'oos

.012
.016
.017
.026
.028
.032
.040

Percent

12.5
18.7
25.0
. 26.5
40.6
43.7
50.0
62.5

Ii6

113

152

120

116.1

116.2

COCCUS FORMS IN CHEDDAR CHEESE.

Several investigators have mentioned coccus forms in relation to
cheese ripening. Von Freudenreich and Thoni x found regularly a
liquefying micrococcus in quite large numbers in fresh Emmental
cheese. They made a number of experimental cheeses, using this
type of organism alone as a starter or together with lactic starters.
They concluded that when present in too great numbers the liquefy-
ing micrococci produced bitterness, but that they disappeared quite
rapidly in practical cheesemaking. Gorini 2 isolated a coccus with
the property of peptonizing casein in an acid medium from Grana,
Emmental, and Edam cheese. He found these forms not only in the
fresh cheese, but in cheese several months old. He thought that
they we're not all of the same type, and he related them to the normal
flora of the udder. In a more recent publication 3 Gorini states that
later investigations have confirmed his opinion that acid-producing
peptonizing ferments are important in the ripening of cooked cheeses
and that the fundamental flora of Grana and other cooked cheeses is
composed of two types of bacteria; (1) lactic acid bacteria, and (2)
acid-producing, peptonizing ferments. In the latter class he in-
cludes bacillus forms with those properties and divers types of cocci.

No reference to coccus forms having been found in considerable

1 Von Freudenreich, Edward, and ThSnl, J. Sur lea bacte"ries du lalt normal et lours
rapports avec la maturation des fromages. Revue Ge'ne'rale du Lait, vol. 2, No. 11, pp.
241-247, Mar. 15 ; No. 12, pp. 271-280, Mar. 30, Lierre, 1903.

* Gorini, C. Sur la presence de bactgries productrices d'acidite" et de pre"sure dans les
fromages en maturation. Rue Ge'ne'rale du Lait, vol. 3, No. 22, pp. 505-510. Lierre,
Aug. 31, 1004.

8 Gorini, C. Studi sulla fabbricazlone del fromaggl Grana, ecc. Italy-Ministero dl
Agricoltura, Industriae Commercia. Bollettino. Anno 9, vol. 1, ser. C, No. 6, pp. 9-17.
Rome, June, 1010.

COCCUS FOKMS IN CHEDDAR CHEESE. 49

numbers has been made in the literature on Cheddar cheese. Hard-
ing and Prucha * report the presence of acid-producing, liquefying
coccus forms in 9 out of 10 cheeses studied. They state that these
forms occurred sufficiently often to suggest that they- might play
some part in the ripening changes, but that they made little headway
in the cheese, and their number, as compared to the total germ con-
tent of the cheese, was relatively insignificant.

Coccus forms which produce a small amount of acidity were oc-
casionally found to be the predominating type of organism in the
cheeses studied, as shown by the milk cultures from high dilutions
of the cheese. Unfortunately, with the dilution method of analysis
this type can be differentiated only when it predominates, for if it
occurs in a culture with the other cheese organisms it can not be
distinguished with certainty from the Bacterium lactis acidi type in
a microscopic preparation, and the titration of the milk gives no
information, since its production of acidity is less than either the
lactic bacilli or Bacterium lactis acidi. However, this type has been
found to predominate at some time or other in 11 of the 13 cheeses
examined by this method.

In 4 of the 11 cheeses the maximum number of coccus forms found
was 100 ; 000,000 per gram, in 4 other cheeses 1,000,000,000, and in the
3 remaining 10,000,000,000 per gram. The time at which they pre-
dominated varied from the 14th to the 161st day. This would indi-
cate that they increase early in the ripening period and maintain
such numbers for a considerable period. The coccus forms have
more or less of a liquefying action on gelatin ; a few show a decided
action, some none at all, while the majority produce a minute de-
pression in the gelatin around the colony. Their action is not com-
parable with that of those organisms usually classed as liquefying
bacteria. They produce small crystals in milk that enables one to
differentiate them from Bacterium lactis acidi. The nature of these
crystals has not yet been determined.

The various cultures produced acidities in milk varying from
0.35 to 0.80 per cent.

As has been previously mentioned, all of the colonies from a cir-
cumscribed area on the lactose-agar plates prepared from the cheeses
last examined were inoculated into milk. The formation of the
crystals was used to distinguish the coccus forms. In some cheeses,
especially those with a low germ content, the cocci have made up
from 10 to 40 per cent of the bacteria as determined by lactose-agar
plate cultures. In other cheeses they have not been detected in many
of the examinations made, and when found were in minor numbers.

1 Harding, H. A., and Prucha, M. J. The bacterial flora of Cheddar cheese. New York
Agricultural Experiment Station, Technical Bulletin 8. Geneva, Dec., 1908. See p. 184.

50 BACTERIOLOGY OF CHEDDAR CHEESE.

It should be mentioned, however, that this method of detecting
the various kinds of organisms in cheese is subject to considerable
error, especially when certain forms are present in much smaller
numbers than other forms, as is the case with the coccus forms as
compared with the lactic bacteria of both groups in many of the
cheeses examined. The error can be reduced by making subcultures
of a greater number of colonies. By the extension of this method
or by the use of some differentiating medium they may be shown to
make up a considerable part of the flora of normal Cheddar cheese.

CHROMOGENIC COCCI.

The chromogenic cocci can be distinguished on the plate cultures
when they are present in appreciable numbers. In a portion of the
work the number of colonies of chromogens appearing on gelatin
plates was determined. The results seemed to indicate a relative
increase late in the ripening period. As has previously been pointed
out, it is impossible to determine, in the case of any group of organ-
isms for which no means of differentiation from other groups has
been found : whether growth is taking place in cheese or not.

It is characteristic of the chromogenic cocci that they persist and
possibly grow under conditions which rapidly destroy less resistant
types. They are present in butter, in which food material is limited,
and in which the moisture represents a saturated solution of sodium
chlorid. Efforts were made to determine their number in cheese by
plating on gelatin containing 3 or 4 per cent of sodium chlorid. The
results were quite satisfactory as far as the chromogenic types were
concerned, but since many other coccus forms did not grow thereon
its use was given up.

In some cheeses very few chromogenic forms have been found,
while in others of similar quality they have been present quite con-
sistently in considerable numbers. In cheese which contained more
than the usual amount of salt they made up a relatively greater pro-
portion of the flora than in other cheese.

LIQUEFYING ORGANISMS.

Attention has been directed to those organisms that show a pro-
nounced liquefying action on gelatin and casein. The results have
been confirmatory of a statement made earlier that they can not be
considered of importance in the ripening of Cheddar cheese since
they are not consistently present in sufficient numbers to exert any
effect.

SEQUENCE IN DEVELOPMENT OF BACTERIAL GROUPS. 51

THE SEQUENCE IN DEVELOPMENT OF BACTERIAL GROUPS IN

CHEDDAR CHEESE.

In the first part of this bulletin it was stated that the normal
ripening of each kind of cheeese is to be looked upon as a problem
in the ecology of microorganisms, and that the only group of organ-
isms which has been found by previous investigators in every
Cheddar cheese in great numbers is the Bacterium, lactis acidi group.
The work herein reported proves that another group of bacteria, the
so-called B. bulgaricus group, develops after the first, and that it
reaches approximately equal numbers.

Each of these groups of organisms produces certain changes in the
cheese mass, consuming the same class of substances and giving out
the same by-products in every cheese which ripens normally. The
second group takes up the work of decomposition at a certain stage
of the ripening, and brings about its own peculiar changes which
prepare the cheese mass for possibly still other types of organisms,
and so on to the end of the ripening.

If a certain sequence of groups of microorganisms is essential for
the preparation of a certain product from raw material, and if the
various members of the sequence find favorable conditions for growth
in the raw material, the resulting product will depend on the first
member of the normal sequence developing to the necessary degree
before the second appears. If the first is overwhelmed by the addi-
tion of great numbers of the second, the decomposition changes will
not be normal. The presence of great numbers of organisms of the
first member of the sequence will not cause a disturbance in the
normal decomposition changes. Thus, in the manufacture of Ched-
dar cheese, cultures of Bacterium lactis acidi are added.

The other essential groups of microorganisms are present in the
milk in small numbers, but as conditions become favorable they de-
velop in the cheese, and a typical Cheddar cheese results.

If heavy inoculations of lactic bacilli are made in milk which con-
tains a small number of Bacterium lactis acidi the normal ecological
balance will be destroyed, and the result will not be a normal product.
Experiments have been made along this line by the use of milk which
has been pasteurized in such a manner that the result has been the
great reduction of all groups of bacteria therein, but the total destruc-
tion of none. In its decomposition such milk is similar to raw milk,
which contains but few bacteria. If cheese is prepared from such
milk after the addition of a culture of Bacterium lactis acidi, it may
ripen in a quite normal manner. The development of the flavor is
usually slower than in a cheese made from raw milk. If to this same
milk is added a culture of lactic bacilli instead of Bacterium lactis
acidi, the ripening is not nearly so normal. The result is an increased
rate of ripening and the production of an abnormal flavor.

52 BACTERIOLOGY OF CHEDDAE CHEESE.

This work also indicates that it is often useless to attempt to
establish the role of any organism in cheese ripening by the addition
of cultures to the milk to be used, since thereby the natural equi-
librium is destroyed, and the results obtained indicate that the addi-
tion has injured the product, and hence the conclusion is drawn that
the organism added is not only not essential, but even harmful,
although the organism may be an essential factor in the decomposi-
tion changes when developing in its normal sequence.

The constant presence in large numbers is the only certain proof
of the importance of an organism or group of organisms in the
ripening of any cheese.

SUMMARY.

1. From the same raw materials various kinds of cheese are pre-
pared, which differ especially in flavor. The factors that determine
whether a cheese to be prepared from a given mass of milk, rennet,
and salt is to be of one kind or another are to be found in the methods
of the cheese maker, who is able to vary in one way or another the
composition of the cheese, with the result that conditions are estab-
lished that favor or retard the growth of the groups of micro-
organisms, which must be the determining factors between different
kinds of cheese.

k 2. The only group of bacteria found constantly in -great numbers
in Cheddar cheese by previous investigators is the Bacterium lactis
acidi group. The functions of this group in Cheddar cheese are,
through their chief by-product, lactic acid

(a) To favor the curdling of milk by rennet.

(5) The bacteria of the milk are held in great part in the curd.
Through the acid they influence the shrinking of the curd and expul-
sion of the whey.

(<?) The acid so changes the nature of the curd as to cause " mat-
ting."

(d) The acid activates the pepsin of the rennet extract.

(e) The acid prevents the growth of putrefactive bacteria in the
cheese.

3. It has been shown that Bacterium lactis acidi is able to form
acid in the absence of the living cell.

4. The development of Bacterium lactis acidi is followed by the
growth of another group of acid-forming bacteria, the Bacillus Bul-
garicus group. They reach numbers comparable with those of the
first group, reaching their maximum numbers within the first month
of the ripening. Since they develop after the fermentation of the
sugar, they must have some other source of carbon and of energy
than milk sugar.

5. It is probable that coccus forms are constantly found in large
numbers in Cheddar cheese.

WAI UBRAflV FACIUTV

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